determination-of-the-activity-of-succinate-nadh-choline-andglyce

Prévia do material em texto

<p>METHODS OF BIOCHEMICAL ANALYSIS VOLUME 22</p><p>Determination of the Activity of</p><p>Succinate. NADH. Choline. and</p><p>a-Glycerophosphate Dehydrogenases</p><p>THOMAS P . SINGER. Department of Biochemistry and Biophysics.</p><p>Universiy af California. San Franckca. Calgarnia. and Molentlar Biology Diuision.</p><p>Yeterans Administration Hospital. San Francisco. California</p><p>I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . 124</p><p>I1 . . . . . 126</p><p>1 . General Considerations . . . . . . . . . . . . . . . . . . . 126</p><p>. . . . . . . . . . . . . . . . . 131</p><p>3 . Succinate-Coenzyme QReductase Assay . . . . . . . . . . . . . 132</p><p>A . Principle . . . . . . . . . . . . . . . . . . . . . 132</p><p>B . Method . . . . . . . . . . . . . . . . . . . . . . 132</p><p>. . . . . . . . . . . . . . . . 133</p><p>B . Method . . . . . . . . . . . . . . . . . . . . . . 133</p><p>C . Comments . . . . . . . . . . . . . . . . . . . . . 136</p><p>5 . Ferricyanide Assay . . . . . . . . . . . . . . . . . . . . 137</p><p>A . Principle . . . . . . . . . . . . . . . . . . . . . 137</p><p>B . Method . . . . . . . . . . . . . . . . . . . . . . 137</p><p>C . Comments . . . . . . . . . . . . . . . . . . . . . 139</p><p>. . . . . . . . . . . . . . . . . 140</p><p>B . Method . . . . . . . . . . . . . . . . . . . . . . 140</p><p>7 . “Reconstitution” Activity . . . . . . . . . . . . . . . . . . 142</p><p>A . Principle . . . . . . . . . . . . . . . . . . . . . 142</p><p>B . Method . . . . . . . . . . . . . . . . . . . . . . 145</p><p>Bound Flavin . . . . . . . . . . . . . . . . . . . . . . 146</p><p>A . Principle . . . . . . . . . . . . . . . . . . . . . 146</p><p>B . Method . . . . . . . . . . . . . . . . . . . . . . 147</p><p>C . Comments . . . . . . . . . . . . . . . . . . . . . 147</p><p>Plants . . . . . . . . . . . . . . . . . . . . . . . . . . 148</p><p>IV . NADH Dehydrogenase and NADH Oxidase Assays . . . . . . . . . . . 151</p><p>1 . General Considerations . . . . . . . . . . . . . . . . . . . 151</p><p>2 . NADH Oxidase Assay . . . . . . . . . . . . . . . . . . . 155</p><p>A . Principle . . . . . . . . . . . . . . . . . . . . . 155</p><p>Assay of Succinate Dehydrogenase and Succinoxidase in Animal Tissues</p><p>2 . Succinoxidase Assay . . . . . . . . . . . . . . . . . . . . 131</p><p>A . Principle and Method</p><p>4 . Phenazine Methosulfate Assay</p><p>A . Principle . . . . . . . . . . . . . . . . . . . . . 133</p><p>6 . Fumarate Reductase Assay</p><p>A . Principle . . . . . . . . . . . . . . . . . . . . . 140</p><p>8 . Determination of Succinate Dehydrogenase by Analysis for Covalently</p><p>111 . Application of Succinate Dehydrogenase Assays to Yeast, Bacteria, and Higher</p><p>B . Method . . . . . . . . . . . . . . . . . . . . . . 155</p><p>123</p><p>Methods of Biochemical Analysis, Volume 22</p><p>Edited by David Glick</p><p>Copyright © 1974 by John Wiley & Sons, Inc.</p><p>124 THOMAS P . SINGER</p><p>3 . NADH-Coenzyme Q Reductase Assay . . . . . . . . . . .</p><p>A . Principle . . . . . . . . . . . . . . . . . . .</p><p>B.Method . . . . . . . . . . . . . , . . . . . .</p><p>C . Comments . . . . . . . . . . . . . . . . . . .</p><p>4 . NADH-Ferricyanide Assay . . . . . . . . . . . . . . .</p><p>B . Method . . . . . . . . . . . . . . . . . . . .</p><p>C . Comments . . . . . . . . . . . . . . . . . . .</p><p>. . . .</p><p>A . Principle . . . . . . . . . . . . . . . . . . .</p><p>B . Method . . . . . . . . . . . . . . . . . . . .</p><p>. C . Comments . . . . . . . . . . . . . . . . . . .</p><p>A . Principle . . . . . . . . . . . . . . . . . . .</p><p>5 . Assay of Low-Molecular-Weight Derivatives of the Enzyme</p><p>6 . Transhydrogenase Assay . . . . . . . . . . . . . . . .</p><p>A . Principle . . . . . . . . . . . . . . . . . . .</p><p>B . Method . . . . . . . . . . . . . . . . . . . .</p><p>. . . . . . . . . . . . . . . . .</p><p>A . Saccharomyccs cercviszat . . . . . . . . . . . . . . .</p><p>B . Candtda utilis . . . . . . . . . . . . . . . . . .</p><p>8 . Determination of NADH Dehydrogenase Content by Piericidin Binding</p><p>V . Assay of Choline Dehydrogenase . . . . . . . . . . . . . . .</p><p>1 . Principle . . . . . . . . . . . . . . . . . . . . .</p><p>2 .Method . . . . . . . . . . . . . . . . . . . . . .</p><p>3 . Comments . . . . . . . . . . . . . . . . . . . . .</p><p>VI . Assay of Mitochondria1 a-Glycerophosphate Dehydrogenase . . . . . .</p><p>1 . Principle . . . . . . . . . . . . . . . . . . . . .</p><p>2 .Method . . . . . . . . . . . . . . . . . . . . . .</p><p>References . . . . . . . . . . . . . . . . . . . . . . . . .</p><p>7 . Application to Yeast</p><p>. .156</p><p>. .156</p><p>. .156</p><p>. .157</p><p>. .158</p><p>. .158</p><p>. .160</p><p>. .161</p><p>. .161</p><p>. .161</p><p>. .163</p><p>. .163</p><p>. .164</p><p>. .164</p><p>. .165</p><p>. .165</p><p>. .165</p><p>. .166</p><p>. . 167</p><p>. .168</p><p>. .168</p><p>. .168</p><p>. .170</p><p>. .170</p><p>. .170</p><p>. .172</p><p>. . 172</p><p>INTRODUCTION</p><p>More than 15 years have elapsed since the publication of the author’s</p><p>previous review in this series on methods of determination of succinate</p><p>dehydrogenase activity (1) . In the intervening years the phenazine metho-</p><p>sulfate (PMS)* assay described in that article has been widely adopted. its</p><p>limitations and the complexities imposed by the unusual regulatory proper-</p><p>ties of succinate dehydrogenase have been recognized. and improved proce-</p><p>dures designed to deal with these problems have been devised . It seems</p><p>appropriate. therefore. to review available information on the determination</p><p>of the activity of the enzyme. as well as its concentration. in various</p><p>biological materials .</p><p>* The following abbreviations are used in this article: CoQ and CoQH2. oxidized and</p><p>reduced forms of coenzyme Q (ubiquinone); CoQdl. CoQ2. etc., homologs of CoQ with one.</p><p>two. etc., isoprenoid units; DCIP. 2. 6.dichlorophenolindophenol. ESP. ETP. ETP, . inner</p><p>membrane preparations. the former two nonphosphorylating. the latter phosphorylating type;</p><p>HEPES. N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; PMS. phenazine methosulfate .</p><p>DETERMINATION OF THE ACTIVITY OF DEHYDROGENASES 125</p><p>In the intervening years a number of other respiratory chain-linked</p><p>flavoproteins have been obtained in purified form. The assay of these in</p><p>membrane-bound and soluble preparations involves theoretical problems</p><p>similar to those which had to be resolved in divising an assay for succinate</p><p>dehydrogenase. Moreover, the assays of some of these enzymes (choline and</p><p>a-glycerophosphate dehydrogenases) are quite similar in principle to the</p><p>spectrophotometric PMS-DCIP procedure for succinate dehydrogenase. For</p><p>these reasons the present article includes also a survey of the determination</p><p>of the activity and content of other respiratory chain-linked flavoproteins.</p><p>The problems inherent in the isolation and reliable assay of flavoproteins</p><p>of the respiratory chain have been reviewed (2,3) and need not be re-</p><p>iterated. Two points, however, deserve emphasis, since failure to appreciate</p><p>them has been the reason for many erroneous and conflicting data in the</p><p>literature concerning the activities, concentrations, and properties of these</p><p>enzymes, as well as for needless polemics concerning these questions.</p><p>First, on extraction of these dehydrogenases from the membrane they</p><p>become very labile, so that, unless great care is taken to minimize or prevent</p><p>modification, the resulting purified enzyme may show activities which are</p><p>quite different from those of the native enzyme. Not only may catalytic</p><p>activity decline markedly during purification but also new activities may be</p><p>created by modification of the protein, as in the cytochrome reductase,</p><p>DCIP reductase, and CoQ reductase activities of the “low-molecular-weight</p><p>form” of NADH dehydrogenase (2). Hence, in order to be certain that a</p><p>given assay method measures the full activity of the enzyme in both soluble</p><p>and particulate preparations, it must be ascertained that no activity has</p><p>been created or unaccountably lost during isolation.</p><p>The second point is the value of the chemical determination of the</p><p>concentration of the enzyme. This approach overcomes problems and un-</p><p>certainties inherent in catalytic assays, such as partial inactivation during</p><p>purification, state of activation of the enzyme, permeability of the</p><p>the enzyme is usually 30 to 40% activated, but</p><p>deactivation increases on freeze-thawing, sonication, and storage (64).</p><p>A special problem in the assay of the dehydrogenase in mitochondria from</p><p>all higher plants studied is the observation (64) that treatment with neither</p><p>Ca2+ nor phospholipase A permits free penetration of the dyes used in the</p><p>assay of succinate dehydrogenase. As a result the apparent succinate de-</p><p>hydrogenase activity may increase as much as two- to threefold on freezing</p><p>overnight or sonication. The best way to assure full penetration of the dyes</p><p>appears to be sonication of the mitochondria. In the author's laboratory the</p><p>mitochondria, suspended in 0.3M mannitol-lmM cysteine, pH 7.2 to 7.4, at</p><p>7 to 8 mg/ml protein concentration, are subjected to two 15-sec sonications,</p><p>with cooling in between, at 0 to 5", with the Bronson S-75 sonifier equipped</p><p>with a microtip probe. This seems to assure full penetration of the dyes, as</p><p>judged by the facts that further sonication or freeze-thawing does not</p><p>increase the measured activity and that the turnover number in preparations</p><p>so treated equals that of mammalian succinate dehydrogenase (64).</p><p>IV. NADH DEHYDROCENASE AND NADH OXIDASE</p><p>ASSAYS</p><p>1. General Considerations</p><p>Although the assay of the respiratory chain-linked NADH dehydrogenase</p><p>in animal tissues is operationally simpler than that of succinate de-</p><p>152 THOMAS P. SINGER</p><p>hydrogenase, the theoretical problems involved are considerably more com-</p><p>plex. Whereas mitochondria contain a single enzyme capable of oxidizing</p><p>succinate, they contain a number of flavoproteins capable of oxidizing</p><p>NADH. This is to be expected from the fact that their prosthetic groups,</p><p>FMN or FAD, in the free form oxidize reduced pyridine nucleotides at</p><p>appreciable rates (65). This potential may be retained, enhanced, or</p><p>suppressed by the environment of the flavin in the holoenzyme. Con-</p><p>sequently, considerable care must be taken to ensure that the assay used</p><p>measures only the activity of the respiratory chain-linked enzyme. This is</p><p>readily ascertained in the assay of the NADH oxidase activity of animal</p><p>tissues by showing that the activity is inhibited by antimycin A, cyanide,</p><p>rotenone, or piericidin A. The problem is more complicated in the case of</p><p>certain yeasts (cf. Section IV.7) and higher plants, which are thought to</p><p>have two respiratory chain-linked NADH dehydrogenases, one dealing with</p><p>intramitochondrial and the other with extramitochondrially generated</p><p>NADH, neither of which is fully rotenone or piericidin or cyanide sensitive</p><p>(66,67).</p><p>In dehydrogenase assays the problem becomes considerably more com-</p><p>plex, largely because of certain unique properties of NADH dehydrogenase.</p><p>This high molecular weight enzyme, in both membrane-bound and soluble</p><p>form, tends to break down to low molecular weight fragments which retain</p><p>catalytic activity on exposure to organic solvents, heat, urea, thiourea,</p><p>proteolytic enzymes, and prolonged contact with the substrate (2,3,68). The</p><p>low molecular weight products arising have altered catalytic properties: the</p><p>substrate specificity and K,,, values are changed, the characteristic high</p><p>reactivity with ferricyanide and the substrate-induced ESR signal of the</p><p>iron-sulfur moieties are lost, and, most importantly, nascent activities not</p><p>seen in the native enzyme arise. Thus transformation to the low molecular</p><p>weight form is accompanied by a very large rise in NADH-DCIP reductase</p><p>(“diaphorase”) activity and the emergence of NADH-cytochrome c reduc-</p><p>tase (antimycin-insensitive) and NADH-CoQ, reductase activities, which</p><p>are not seen in unmodified preparations (69-71). Disappearance of the ESR</p><p>signal at g = 1.94 and of the ferricyanide activity is due to loss of the</p><p>iron-sulfur moieties, while the appearance of new activities seems to be the</p><p>result of conformation changes around the flavin site, exposing the flavin,</p><p>which permits direct reoxidation of the reduced flavin by CoQ, cytochrome</p><p>c, DCIP, and ferricyanide. [The relatively low NADH-Fe(CN),-3 reductase</p><p>activity of damaged preparations may also be due to direct interaction with</p><p>the flavin, since it differs in several important respects (56) from the</p><p>ferricyanide reductase activity of the high-molecular-weight form, which</p><p>proceeds via iron-sulfur center 1 of the enzyme (72,73).]</p><p>Before these facts were appreciated, the extraction procedures used for</p><p>DETERMINATION OF THE ACTIVITY OF DEHYDROGENASES 153</p><p>isolation of this enzyme had been the very ones which cause breakdown to</p><p>the low molecular weight form (heat, heat-acid-ethanol, urea, thiourea, etc.)</p><p>Not unexpectedly a number of preparations from the same tissue, bearing</p><p>different names, were described in the literature. When the first purified</p><p>preparation of the high molecular weight form was obtained under mild</p><p>conditions of extraction (74), it could be readily shown that exposure to the</p><p>agents used in previous attempts to isolate the enzyme results in breakdown</p><p>to low molecular weight fragments identical in molecular and catalytic</p><p>properties to those directly extracted from tissues by these methods</p><p>(69,70,75).</p><p>An interesting instance is the preparation called “NADH-ubiquinone</p><p>reductase” (76). This was extracted under conditions (heat-acid pH-ethanol)</p><p>substantially identical to those used in the isolation of NADH-cytochrome c</p><p>reductase many years before (77), but was stated to be both different from</p><p>the latter and the most nearly physiological form of the enzyme, because it</p><p>catalyzed the reduction of long-chain coenzyme Q derivatives (CoQ,,</p><p>CoQ,,) and was inhibited by rotenone and amytal. (The latter compounds</p><p>interrupt electron flux between NADH dehydrogenase and CoQ,, in mi-</p><p>tochondria.) Careful comparison of the molecular and catalytic criteria,</p><p>however, revealed no significant difference between NADH-ubiquinone and</p><p>NADH-cytochrome c reductase, and the absence of rotenone-sensitive</p><p>CoQl0 reduction was traced to the use of aged particles and a lyophilization</p><p>step in the preparation of the latter, both of which result in decay of CoQ,,</p><p>reductase but not of cytochrome reductase activity (78). Moreover, the CoQ</p><p>reductase activity and sensitivity to amytal and rotenone turned out to be</p><p>artifactual, the former because it did not require lipids, the latter because it</p><p>was only a partial inhibition, relieved on increasing the concentration of the</p><p>inhibitor (75,78). In fact, exposure of the high molecular weight form of</p><p>NADH dehydrogenase (which cannot react with either CoQ, or rotenone</p><p>because these reactions require lipids which are removed during isolation) to</p><p>the conditions used in the extraction of NADH-ubiquinone reductase re-</p><p>sulted in breakdown to the low molecular weight form, with emergence of</p><p>partially rotenone- and amytal-sensitive CoQl0 reductase activity (Table</p><p>11). Thus reliance on “physiological” criteria-in this case reaction of the</p><p>dehydrogenase with CoQ,, and sensitivity to rotenone-may yield mislead-</p><p>ing results unless care is taken to ensure that the activity or reaction in</p><p>question occurs in the same manner in the isolated enzyme as in intact</p><p>particles.</p><p>As a result of investigations in many laboratories, most workers agree that</p><p>in mammalian preparations the ferricyanide assay (Section IV.4), under the</p><p>conditions described, provides the most reliable measure of NADH de-</p><p>hydrogenase activity. Despite the presence of numerous flavoproteins in</p><p>154 THOMAS P. SINGER</p><p>TABLE I1</p><p>Conveision of NADH Dehydmgenase to NADH-CoQ Reductase</p><p>Highly purified NADH dchydrogenase, &solved in 0.05M phoephate, pH</p><p>7.6, was treated at pH 5.3, 43’, in 9% ethanol as in the extraction of</p><p>“NADH-ubiquinone reductax.” The suspension was chilled, neutralized</p><p>to pH 6.8, and precipitated protein was removed by centrifugation. From</p><p>Mach et al. (75).</p><p>Units Specific</p><p>activity</p><p>Before After Before After</p><p>conversion” conversionb conversion* conversionb</p><p>Fe(CN),-3 471,000 8185 47</p><p>1 138</p><p>Cytochrome t reductase 92 3176 0.092 53.6</p><p>46 reductase 22 900 0.22 15.2</p><p>pI reductase 279 1771 0.28 29.9</p><p>Qo reductase 16 720 0.016 12.2</p><p>“Total protein, lo00 mg.</p><p>bTotal protein, 5.9 mg.</p><p>mitochondria, potentially or actually capable of oxidizing NADH, with this</p><p>assay virtually all the activity in inverted inner membrane preparations</p><p>(ETP, ETP,), in fragments of the respiratory chain (Complex I), and in</p><p>phospholipase-extracted, soluble ones is ascribable to NADH dehydrogenase.</p><p>Furthermore, the same turnover number is obtained in the NADH-</p><p>Fe(CN),-3 assay and in studies of the rate of reduction of iron-sulhr center</p><p>1 of the enzyme by ESR techniques, suggesting that the full activity of the</p><p>enzyme is being measured (79).</p><p>There is at present no satisfactory way to measure either NADH de-</p><p>hydrogenase or oxidase activity in intact mitochondria, since the enzyme</p><p>appears to be localized on the inside of the inner membrane and neither</p><p>NADH nor ferricyanide can freely cross the membrane. Measurement of the</p><p>rate of oxidation of NAD-linked substrates in intact mitochondria is not an</p><p>adequate substitute, since the activity of the NAD-linked dehydrogenases,</p><p>rather than that of the flavoprotein, is likely to be rate limiting even in the</p><p>absence of respiratory control. Application of the ferricyanide assay to</p><p>preparations from higher plants is complicated by the apparent existence of</p><p>dual NADH dehydrogenases. Current studies in the author’s laboratory</p><p>(80), however, suggest that, with minor modifications, the method may be</p><p>useful for the measurement of the activity of the “external” NADH de-</p><p>hydrogenase.</p><p>DETERMINATION OF THE ACTIVITY OF DEHYDROGENASES 155</p><p>2. NADH Oxidase Assay</p><p>A. PRINCIPLE</p><p>The NADH oxidase activity of undamaged mammalian mitochondria</p><p>cannot be measured because NADH cannot cross the inner membrane. In</p><p>inverted submitochondrial particles NADH oxidase may be assayed either</p><p>spectrophotometrically or polarographically. Although the former method is</p><p>widely used, care must be taken to ensure that the response of the</p><p>spectrophotometer is linear with increasing NADH concentration in the</p><p>range to be used. There are statements in the literature that NADH, at</p><p>concentrations in excess of the level required for saturation (about 0.15mM)</p><p>inhibits the oxidase activity. Such observations may have been due to</p><p>nonlinear response at 340 nm of the spectrophotometers used, since in the</p><p>author’s laboratory the NADH oxidase activity of ETP did not vary between</p><p>0.15 and 1mM NADH concentrations in polarographic assays, while with</p><p>the Gilford 220 spectrophotometer the rate appeared to decline above</p><p>0.4mM NADH (81).</p><p>In preparations of closed vesicles (ETP, ETP,, ESP) the addition of</p><p>cytochrome c is not required; in fact, added cytochrome c hardly reacts with</p><p>the NADH oxidase system. In lyophilized, organic solvent-extracted, or</p><p>deoxycholate-treated preparations added cytochrome c stimulates the oxi-</p><p>dase activity. The optimal level of cytochrome c depends on the type of</p><p>preparation and should be determined in each case; however, it is usually in</p><p>the range used for succinoxidase assays (Section 11.2).</p><p>B. METHOD</p><p>REAGENTS. Potassium phosphate buffer, 0.08 to 0.1 M , containing 50pM</p><p>EDTA, pH 7.4.</p><p>NADH, 0.01 M , neutral solution, freshly prepared each day.</p><p>Spectrophotometric Procedure. The spectrophotometer cuvette, pre-</p><p>viously equilibrated at 30°, containing 0.05 ml of 0.01M NADH and</p><p>sufficient buffer to give 3.0 ml final volume, is placed in a thermostated</p><p>recording spectrophotometer. The reaction is started by the addition of</p><p>enzyme (usually 0.01 to 0.025 ml), and the decrease in absorbance at 340</p><p>nm is recorded. Activity is calculated from the molar absorbance of NADH</p><p>(6.22 x lo3).</p><p>Polarographic Procedure. The same reaction mixture is used as in the</p><p>spectrophotometric method, but the volume of reactants is adjusted to the</p><p>volume of the oxygen electrode chamber. Some investigators prefer to use</p><p>156 THOMAS P. SINGER</p><p>final NADH concentrations as high as ImM, although the activity usually</p><p>does not increase above 0.15mM NADH.</p><p>3. NADH-Coenzyme Q Reductase Assay</p><p>A. PRINCIPLE</p><p>The assay of NADH-CoQ reductase activity with water-soluble, short-</p><p>chain CoQ homologs (CoQ, or CoQJ is relatively straightforward, but</p><p>these compounds are not commercially available at present. With long-chain</p><p>homologs (CoQ, or the physiological quinone, CoQ,,), which are available,</p><p>the procedure becomes exceedingly complicated because of the insolubility</p><p>of the quinone in aqueous solutions. The procedure given below is essentially</p><p>that of Pharo et al. (76) and is based on following the disappearance of</p><p>NADH spectrophotometrically at 340 nm. Either C o Q or CoQ,, is added</p><p>as a methanolic solution to the complete reaction mixture (except enzyme)</p><p>and incubated for a carefully controlled time interval at 30". During this</p><p>interval the bulk of the added C o Q precipitates, so that during the brief</p><p>assay period initiated by the addition of the enzyme little additional</p><p>turbidity change occurs. To correct for the slight turbidity change occurring</p><p>during assay the latter is carried out in a double-beam or dual-</p><p>monochromator instrument, such as the Gary 14 or the Aminco-Chance</p><p>spectrophotometer, with water, instead of NADH, added to the other</p><p>components in the reference cuvette. (Alternatively, a single-beam instru-</p><p>ment equipped with an automatic cuvette changer may be used to permit</p><p>concurrent recording of the rates in the experimental and blank cuvetts.) If</p><p>none of these instruments is available, with soluble enzyme samples a stand-</p><p>ard recording spectrophotometer gives satisfactory results if the experimental</p><p>and blank samples are assayed in succession.</p><p>B. METHOD</p><p>REAGENTS. Tris-sulfate buffer, 0.1 M, pH 8.0, at 30".</p><p>NADH, 4mM in 0.1 M Tris-sulfate, pH 8.0, prepared daily.</p><p>KCN, 30mM, neutralized to pH-8, prepared fresh every</p><p>other day, preserved at 0" when not in use.</p><p>CoQ, or CoQ,, 3mM solution in 0.1 M Tris-sulfate, pH 8.0.</p><p>Preserved in freezer, protected from light.</p><p>CoQs, 6mM, or CoQ,,, 4mM in methanol. CoQ,,must be</p><p>heated to 50" to go into solution and maintained at this</p><p>temperature while pipetting. Both solutions are stored in the</p><p>freezer, protected from light.</p><p>Procedure for CoQ, and CoQ,. An aliquot of the enzyme, buffer (to give</p><p>a final volume of 1 ml during assay), and 0.1 ml cyanide are equilibrated for</p><p>DETERMINATION OF THE ACTIVITY OF DEHYDROGENASES 157</p><p>min at 30" in a stoppered cuvette and then placed in the thermostated cell</p><p>compartment (at 30") of a recording spectrophotometer. NADH (0.30 ml)</p><p>is added and the absorbance at 340 nm is recorded for a few seconds to see</p><p>whether any oxidation occurs without CoQ. The reaction is then started by</p><p>adding 30 pl of CoQ, or CoQ,, and the rate is calculated as the difference</p><p>in the decrease in absorbance at 340 nm with and without the quinone. The</p><p>blank rate should be negligible, but in no event over 10% of the rate with</p><p>CoQ.</p><p>Procedure for CoQ,. Water, Tris-sulfate buffer, C O G , and KCN stock</p><p>solutions are brought to 30" in test tubes. They are pipetted into a pair of</p><p>spectrophotometer cells in the following order: H,O (1.25 ml), Tris-sulfate</p><p>(1.5 ml), CoQ, (0.05 ml), and KCN (0.1 ml). The cell contents are mixed</p><p>after the addition of C O G and also after the KCN. The two cuvettes are</p><p>incubated for exactly 2 rnin at 30" and transferred to the cell compartment</p><p>of the double-beam spectrophotometer (thermostated at 30°), and 0.1 ml of</p><p>NADH is added to one (experimental), 0.1 ml H,O to the other (reference).</p><p>Exactly 3 min after the addition of KCN the reaction is initiated by the</p><p>addition of enzyme in a small volume (0.002 to 0.004 ml). The decrease in</p><p>absorbance at 340 nm is then recorded for a short period (30 to 45 sec), and</p><p>the rate is determined from the initial, linear part of the curve. With the</p><p>Cary 14 spectrophometer the chart speed</p><p>recommended is 3 in./min and the</p><p>absorbance range 0 to 0.5 and 0.5 to 1.0, with the range switch of the</p><p>recorder on automatic.</p><p>With soluble enzyme samples the procedure above may be simplified by</p><p>omitting the cyanide and adding the NADH immediately after the CoQ,</p><p>incubating 2.5 min at 30", and initiating the reaction by the addition of</p><p>enzyme at 3 min. As noted above, with soluble enzymes the experimental</p><p>and reference (blank) samples may be run in succession, if the timing is</p><p>rigidly controlled.</p><p>Procedure for CoQ,,. With CoQo the same procedure is followed, except</p><p>that the quinone solution (and the micropitette used for delivery) is</p><p>maintained and pipetted at 50". Because of rapid evaporation of methanol</p><p>at this temperature, the concentration of CoQ, must be periodically de-</p><p>termined by dilution in methanol, using the molar extinction at 275 nm</p><p>= 1 4 . 6 ~ lo3.</p><p>C. COMMENTS</p><p>The NADH-CoQ reductase activity of membranal or other particulate</p><p>preparations is only a small fraction of the NADH-ferricyanide activity,</p><p>because in the former determination reoxidation of NADH dehydrogenase</p><p>by the added quinone is rate limiting, whereas in the latter the reduction of</p><p>158 THOMAS P. SINGER</p><p>the enzyme by NADH is the rate-limiting step. Soluble preparations of the</p><p>high molecular weight form of the enzyme do not reduce CoQ homologs a t</p><p>appreciable rates because the lipids needed for this interaction, including</p><p>endogenous CoQ,,, are removed during extraction. However, CoQ reduc-</p><p>tase activity may appear after various treatments of the purified enzyme,</p><p>such as exposure to 38", which elicits relatively high CoQ, reductase activity</p><p>(71) (cf. Section IV.1). Such artifactual reduction of CoQ homologs may be</p><p>distinguished from the reaction as seen in intact particles by the fact that</p><p>they are either not inhibited by amytal and rotenone (reduction of CoQ,</p><p>and CoQ2) or only partially inhibited ( C O G , CoQ,,), the inhibition being</p><p>reversed by excess rotenone (75).</p><p>4. NADH-Ferricyanide Assay</p><p>A. PRINCIPLE</p><p>The best method available for measurement of the activity of the re-</p><p>spiratory chain-linked NADH dehydrogenase is assay of the rate of reduc-</p><p>tion of ferricyanide. Since ferricyanide has two reaction sites in the NADH</p><p>oxidase system and thus double reciprocal plots may be biphasic (Figure 9),</p><p>and since the K, for ferricyanide may change on extraction and purification</p><p>of the enzyme, activity should be expressed at infinite concentration of the</p><p>I5 t</p><p>10 I .A</p><p>/ X-</p><p>X'</p><p>I'</p><p>1 1 I I I</p><p>Figure 9. Variation of the NADH dehydrogenase activity of ETP with ferricyanide concentra-</p><p>tion. Routine assay conditions, with 0.07 mg of ETP (specific activity=25) and 1.8 amoles of</p><p>NADH in 3 ml total volume. Abscissa, reciprocal ferricyanide concentration, in milliliters of</p><p>IO-'M ferricyanide present in 3 ml final volume. Ordinate, reciprocal optical density change</p><p>per minute at 420 nm. With 0.3 and 0.4 rnl of femcyanide present, the assays were conducted</p><p>at 440 nm, and with 0.5 to 1.0 ml of ferricyanide at 450 nm and were then calculated for 420</p><p>nm. X-X, control; 0 4 , with 1.5 X 10a7M antimycin or 3 X 10-3M Amytal present; 0</p><p>-0, with I X lOP3M cyanide or 6.6X lO-'M azide present. From Minakami et at. (82).</p><p>DETERMINATION OF THE ACTIVITY OF DEHYDROGENASES</p><p>FIXED F~ ( c N ) ~</p><p>ASSAY</p><p>I</p><p>r'</p><p>159</p><p>15 t</p><p>10 I</p><p>I x'</p><p>Figure 10. Competitive inhibition of soluble dehydrogenase in the ferricyanide assay by high</p><p>NADH concentrations. Conditions as in Figure 9. U, 1.5X10-4M NADH; 0-0,</p><p>3 X 10-4M NADH; X-X, 6 X lO-*M NADH. From Minakami et al. (82).</p><p>oxidant [ V,, (ferricyanide)]. Another reason why reliable activity mea-</p><p>surements with this enzyme must be based on extrapolation to infinite</p><p>oxidant concentration is that NADH and other reduced pyridine nucleotide</p><p>analogs, as well as NAD, the product, inhibit competitively at high con-</p><p>centrations (Figure 10). It has been suggested that NADH at high con-</p><p>centrations binds at a second site on the flavoprotein which is either also the</p><p>combining site of ferricyanide or is close enough to it to interfere with</p><p>reoxidation of the enzyme by ferricyanide. Thus, while at fixed ferricyanide</p><p>concentrations NADH in excess of 1.5 X lOP4M is inhibitory, at V,,, the</p><p>inhibition is not noted (Figure 11).</p><p>RATE</p><p>6 , I I I</p><p>4 8 12 16</p><p>DPNH CONC. (,lo4)</p><p>Figure 11. Variation of NADH dehydrogenase activity with NADH concentration in the</p><p>ferricyanide assay. Upper curve, V,, (ferricyanide) values; lower curve, activity at 8.3X</p><p>10-4M ferricyanide concentration. Conditions were as in the method of Section IV.4, with 0.09</p><p>mg of ETP (protein basis) in each cuvette. Modified from Minakami et al. (82).</p><p>1 60 THOMAS P. SINGER</p><p>In the original version of the ferricyanide assay (82) phosphate buffer at</p><p>pH 7.4 was used. Inorganic phosphate is strongly complexed by the enzyme,</p><p>imparting to it some unusual properties (83). In the phosphate complex the</p><p>enzyme tends to show increasingly steep slopes in double reciprocal plots as</p><p>the pH increased, so that at the optimum (pH 7.8 at 30") the curve is too</p><p>steep for accurate extrapolation. In the revised procedure (84) given below,</p><p>this difficulty is overcome by substituting triethanolamine buffer, pH 7.8,</p><p>which yields reasonable slopes.</p><p>An important consideration in the assay of the dehydrogenase is that the</p><p>temperature should not exceed 30°, particularly with soluble preparations,</p><p>because above this temperature modification of the protein occurs rapidly</p><p>With the precautions mentioned, the ferricyanide assay appears to mea-</p><p>sure the full activity of the native, high molecular weight form of the</p><p>dehydrogenase, as judged by the fact that the rates of oxidation of various</p><p>reduced pyridine nucleotides by ferricyanide and the rates of reduction of</p><p>iron-sulfur center 1 of the enzyme, monitored by EPR at g=1.94, are in</p><p>good agreement (79). The assay works equally well in particulate and in</p><p>soluble preparations, so that no activity is either created or lost on mild</p><p>extraction (74). In intact mammalian mitochondria the method cannot be</p><p>used because ferricyanide does not cross the inner membrane.</p><p>Although on fragmentation of the enzyme by heat or heat-acid-ethanol</p><p>(Table II), or by other methods, reactivity with ferricyanide is usually the</p><p>first catalytic property lost, the decay is incomplete. Hence, after complete</p><p>conversion to the low molecular weight species 2 to 3% of the activity may</p><p>remain, although the EPR-active iron-sulfur center 1 , which is thought to be</p><p>the reaction site of ferricyanide in the native enzyme (68), is no longer</p><p>detectable. This suggests that the ferricyanide reacts at a different site in the</p><p>low molecular weight form of the enzyme; in fact, it appears likely that in</p><p>such preparations ferricyanide reoxidizes the flavin component directly (68).</p><p>The characteristics of the NADH-ferricyanide activity in such preparations</p><p>are markedly changed: high phosphate concentrations do not inhibit the</p><p>enzyme, and no competitive inhibition is evident on adding excess substrate</p><p>(70).</p><p>(85).</p><p>B. METHOD</p><p>REAGENTS. Triethanolamine buffer, 0.1 2M, pH 7.8 at 30".</p><p>K,Fe(CN), in H,O, 0.01M, preserved in amber bottle.</p><p>P-NADH, about 9mM in 0.1% (w/v) KHCO,, prepared</p><p>daily.</p><p>Procedure. To a series of six spectrophotometer cuvettes of 1-cm light</p><p>path there are added 1 ml buffer, H,O to give a final volume of 3 ml during</p><p>DETERMINATION OF THE ACTIVITY OF DEHYDROGENASES 161</p><p>assay, and varying amounts of ferricyanide. The amounts of oxidant recom-</p><p>mended for accurate determination of V,,, are 0.5, 0.2, 0.125, 0.08, 0.06,</p><p>and 0.05 ml. Since at the lowest two concentrations the experimental error</p><p>tends to be greater, some workers prefer to use a narrower range of</p><p>ferricyanide concentrations: 0.5, 0.2, 0.125, 0.1, 0.08, and 0.07 ml. The</p><p>cuvettes are incubated in a water bath at 30" and transferred, one at a time,</p><p>to the cell compartment of a recording</p><p>spectrophotometer, which is ther-</p><p>mostated at 30". Next, NADH solution (0.05 ml) is added and the absor-</p><p>bance decrease at 420 nm is recorded for 15 to 30 sec. This is the blank rate.</p><p>Enzyme is then added in a small volume (5 to 100 PI) to start the reaction,</p><p>and the absorbance decline is again recorded. From the enzymatic rate</p><p>(initial slope) the blank rate is subtracted and the activity is calculated by</p><p>plotting reciprocal absorbance change per minute against reciprocal volume</p><p>of ferricyanide and extrapolating to V,, (ferricyanide). For conversion to</p><p>absolute units the molar extinction coefficient of the oxidant is taken as</p><p>1 X lo3, keeping in mind that 1 mole of NADH corresponds to 2 moles of</p><p>ferricyanide.</p><p>C. COMMENTS</p><p>Perhaps the most important limitation on the ease and accuracy of the</p><p>method is the type of spectrophotometer used. In addition to the obvious</p><p>requirement of stability, the instrument should have features permitting</p><p>instant change of chart speed, absorbance range, and absorbance offset. This</p><p>allows adjustment of the absorbance range and chart speed within the first 2</p><p>to 3 sec after mixing, so that the tracing approximates a 45" angle. With the</p><p>Gilford model 220 instrument and a Texas Servoriter I1 (pushbutton trans-</p><p>mission change of chart speed in the range of 1.5, 3, 6, 12 and 24 in./min.)</p><p>used in this laboratory, samples varying 50- to 100-fold in NADH de-</p><p>hydrogenase activity may be assayed without dilution or the taking of</p><p>different aliquots. In this laboratcry the absorbance range used at the higher</p><p>ferricyanide concentrations is 0.5, with a chart speed of 6 to 12 in./min, and</p><p>at lower ones 0.2, with a chart speed of 3 to 6 in./min. Some investigators</p><p>prefer to use a constant range of 0.2 absorbance unit at all ferricyanide</p><p>concentrations and vary only the chart speed.</p><p>With some practice the entire determination may be completed in 20 to</p><p>25 min, with an accuracy of ?5%.</p><p>5. Assay of Low Molecular Weight Derivatives of the</p><p>Enzyme</p><p>A. PRINCIPLE</p><p>The low molecular weight form of the enzyme may be assayed by</p><p>following the reduction of ferricyanide, DCIP, or cytochrome c. As discussed</p><p>162 THOMAS P. SINGER</p><p>.TRANSHYDR.</p><p>a</p><p>10</p><p>></p><p>t</p><p>L</p><p>t-</p><p>2</p><p>HOURS AT 2 2 ' C</p><p>Figure 12. Time course of degradation of highly purified NADH dehydrogenase with crystalline</p><p>subtilisin. Four milliliters of NADH dehydrogenase solution (1 1.8 mg protein/ml) were</p><p>incubated at 22" in 0.01M phosphate, pH 7.4, with 1.18 mg subtilisin. From Cremona et al.</p><p>(70).</p><p>in the preceding section, the ferricyanide reductase activity of this form of</p><p>the enzyme represents a different reaction mechanism from that observed in</p><p>the high molecular weight form. The latter does not reduce cytochrome c</p><p>directly at appreciable rate and has only a relatively small DCIP reductase</p><p>activity. These activities emerge on conversion to the low molecular weight</p><p>form by a variety of agents, but not at the same rate (Figures 12 and 13).</p><p>2000</p><p>I500</p><p>15 30 45 60 10 20 30</p><p>MINUTES</p><p>Figure 13. Time course of degradation of the high molecular weight form of NADH de-</p><p>hydrogenase with urea. The purified enzyme was incubated at 22" in 0.05M Na arsenate</p><p>buffer, pH 7.5, containing urea at the indicated concentration, at 7.5 mg proteinjml. From</p><p>Cremona et al. (70).</p><p>DETERMINATION OF THE ACTIVITY OF DEHYDROGENASES 163</p><p>Consequently, the relative activity ratios of the low molecular weight form in</p><p>these three assays depend on the method of preparation. Another reason for</p><p>this behavior is that the flavin is readily lost from the low molecular weight</p><p>form (69), and this loss is reflected by a lesser decline in ferricyanide</p><p>reductase than in DCIP or cytochrome reductase activities (86).</p><p>B. METHOD</p><p>REAGENTS. Triethanolamine buffer, 0.3A4, pH 8.5 (at 0').</p><p>P-NADH, 0.006M in 0.1% (w/v) KHCO,, prepared fresh</p><p>Cytochrome c (from horse heart), 1% (w/v), in H,O, pre-</p><p>DCIP, 0.05% (w/v) in 0.1.44 triethanolamine buffer, pH 8.5</p><p>daily.</p><p>served in freezer.</p><p>(cf. Section 11.4).</p><p>Procedure. Each of a series of six spectrophotometer cuvettes of 1-cm light</p><p>path receives 0.2 ml of buffer, 0.1 ml of NADH solution, and water to give a</p><p>final volume of 3 ml during assay. The volume of cytochrome c solution</p><p>added is varied between 0.05 and 0.5 ml (0.05, 0.07, 0.10, 0.125, 0.2, and 0.5</p><p>ml). After 3-min equilibration at 30" the cuvettes are placed in the cell</p><p>compartment of the recording spectrophotometer, thermostated at 30', and</p><p>the reaction is initiated by the addition of enzyme in a small volume (0.01 to</p><p>0.05 ml). The initial rate of absorbance decrease at 550 nm is recorded, and</p><p>the results are calculated from double reciprocal plots by extrapolation to</p><p>infinite cytochrome c concentration [ V,,, (cyt. c) ] , using the differential</p><p>molar extinction coefficient, 19.1 X lo3. Note that the oxidation of 1</p><p>mole of NADH reduces 2 moles of cytochrome c.</p><p>In the measurement of DCIP reductase activity the same procedure is</p><p>followed, but the dye concentration is varied between 1 X lop5 and 8X</p><p>l O P 5 A 4 and the absorbance decrease is followed at 600 nm. The molar</p><p>extinction coefficient is again 19.1 X lo3, but 1 mole of NADH reduces 1</p><p>mole of DCIP.</p><p>C. COMMENTS</p><p>Triethanolamine buffer is used in lieu of 2-amino-2-methyl-l,3-</p><p>propanediol at the same pH (77) in the cytochrome reductase assay because</p><p>the slope in double reciprocal plots tends to be less steep in the former buffer</p><p>(69). Recording speeds of 10 to 12 in./min are recommended at all con-</p><p>centrations of cytochrome c because the measured rate declines rapidly</p><p>(Figure 14), so that strict proportionality between observed rate and enzyme</p><p>concentration is obtained only if initial rates are measured (initial 10 to 15</p><p>sec). Scale expansion (absorbance range) must be adjusted to compensate for</p><p>the variation of observed rate with concentration of cytochrome c.</p><p>164</p><p>E550</p><p>THOMAS P. SINGER</p><p>ENZ. SPEC.ACT: A. B.</p><p>0.- Z" : 52 ' 52</p><p>30."- 60.' : 23 14</p><p>30:-90." : I9 10</p><p>7 f EN2</p><p>147 EN2</p><p>I 0 I I L I . I</p><p>6. Transhydrogenase Assay</p><p>A. PRINCIPLE</p><p>NADH dehydrogenase catalyzes transhydrogenations between various</p><p>pyridine nucleotides (84). The most convenient reaction for assay purposes is</p><p>the transhydrogenation between NADH and acetylpyridine adenine dinuc-</p><p>leotide (AcPyAD):</p><p>NADH + AcPyAD+NAD+ + AcPyADH</p><p>The reaction may be followed spectrophotometrically by taking advantage</p><p>of the differences in the absorption spectra of NADH and of the reduced</p><p>form of AcPyAD (88). At 375 nm the difference in molar extinction</p><p>coefficients, cAcfiADH - eNADH, equals 5.1 X lo3. The measured rate does not</p><p>seem to vary with NADH concentration in the range of 1X10-4M and</p><p>5 X 10-4M but varies significantly with the concentration of AcPyAD.</p><p>Hence the rate of transhydrogenation is measured keeping the concentration</p><p>of NADH (1.5 X lO-*M) fixed but varying the amount of AcPyAD and</p><p>extrapolating to V,, with respect to the latter.</p><p>Only a small fraction of the "not-energy-linked'' transhydrogenase activ-</p><p>ity of mitochondria and only about one-third of the reaction in ETP appears</p><p>to be ascribable to the respiratory chain-linked NADH dehydrogenase (84).</p><p>Hence this assay is not recommended for membranal preparations.</p><p>DETERMINATION OF THE ACTIVITY OF DEHYDROGENASES 165</p><p>B. METHOD</p><p>REAGENTS. Triethanolamine buffer, 0.12A4, pH 7.8, at 30".</p><p>NADH, 0.01A4, neutral, prepared fresh daily.</p><p>AcPyAD, 1.5 X 10-3M (Pabst Laboratories). The concentra-</p><p>tion of the solution is checked by its absorbance at 260 nm</p><p>(cZm= 16.4X lo3).</p><p>Procedure. To a series of five spectrophotometer cuvettes, 1-cm light path</p><p>and 1-ml volume, are added 0.4 ml of buffer, 0.015 ml of NADH, water to</p><p>give a final volume of 1 ml during assay, and varying volumes of AcPyAD</p><p>solution (0.1, 0.2, 0.3, 0.4, and 0.5 ml). After 3-min equilibration at 30", the</p><p>cuvettes are placed in the thermostated (at 30") cell compartment of a</p><p>recording spectrophotometer. The</p><p>reaction is initiated by addition of the</p><p>enzyme in a small volume (0.01 to 0.025 ml), and the initial rate of</p><p>absorbance change at 375 nm is recorded. The results are calculated by</p><p>plotting reciprocal rate of absorbance change against reciprocal volume of</p><p>AcPyAD used and extrapolating to V,,, using the differential extinction</p><p>coefficient given under "Principle."</p><p>7. Application to Yeast</p><p>A. SACCHAROMYCES CEREVISIAE</p><p>The specificity of NADH dehydrogenase in baker's yeast mitochondria</p><p>and submitochondrial particles for electron acceptors is significantly</p><p>different from that of the mammalian enzyme (56,89). Ferricyanide does not</p><p>react sufficiently fast to measure the full activity; DCIP reacts faster, but</p><p>short-chain CoQ homologs are the best electron acceptors. As in heart</p><p>particles, the curve relating reciprocal activity to reciprocal ferricyanide</p><p>concentration is biphasic, suggesting the existence of two reaction sites</p><p>(Figure 15). In contrast to the heart enzyme, however, the inclusion of</p><p>antimycin to abolish electron flux from the dehydrogenase to the second site</p><p>(cytochrome c- cl) shows that the reaction of the flavoprotein itself with</p><p>ferricyanide is relatively slow (cf. Figures 9 and 15). The main objection to</p><p>the use of DCIP with yeast preparations is that it is not specific for</p><p>mitochondria1 NADH dehydrogenase, since both anaerobic cells and mu-</p><p>tants devoid of mitochondrion show appreciable NADH-DCIP reductase</p><p>activity (56,89).</p><p>The activity of NADH oxidase is measured polarographically, essentially</p><p>as described in Section IV.2, but the buffer concentration is 0.04M, the</p><p>temperature is 25", and the inclusion of heart muscle cytochrome c (about</p><p>0.2 mg/ml) is essential for full activity with both submitochondrial particles</p><p>and mitochondria isolated by means of Nossal or Braun shakers (56).</p><p>166 THOMAS P. SINGER</p><p>0-</p><p>€3-</p><p>ANf/MYC/#</p><p>/ =</p><p>6 -</p><p>" .. - .1</p><p>-/</p><p>N O ANT/MYC/N</p><p>I I I 1</p><p>I 2 3 4</p><p>I</p><p>m M Fe (CN);</p><p>Figure 15. Spectrophotometric assay of the NADH-Fe(CN)L3 reaction at 259 Abscissa, recipro-</p><p>cal concentration of oxidant as milliliters of lO-*M ferricyanide; ordinate, reciprocal activity.</p><p>An ETP preparation was used in 0.06M triethanolamine buffer (pH 7.8). The cuvettes</p><p>contained 3.3mM azide and, where indicated, 1 pA4 antimycin. From Singer et al. (56).</p><p>The reduction of CoQ is assayed as in the method of Section IV.3, with</p><p>the following changes: the temperature is 25", the concentrations of NADH</p><p>and C o Q are 0.125mM and 0.2mM, respectively; 0.15 mg of Asolectin is</p><p>included in the reaction, and with particles 2mM azide and lOP6M antimy-</p><p>cin A are also added.</p><p>B. CANDIDA UTILIS</p><p>Unlike animal tissues and S. cereviszae but like higher plants, C. utilis</p><p>mitochondria are thought to have dual NADH dehydrogenases, both linked</p><p>to the respiratory chain, a piericidin-sensitive one concerned with NADH</p><p>produced in the matrix and a piericidin-insensitive one concerned with</p><p>NADH produced outside of the inner membrane (90). The evidence for two</p><p>enzymes is indirect, for only one enzyme has been studied extensively in</p><p>submitochondrial particles (ETP) (88) and partially purified preparations</p><p>(91). Since this enzyme reacts directly with NADH and its coupling to the</p><p>respiratory chain is subject to piericidin inhibition, it is usually assumed that</p><p>the enzyme originates from the inside of the inner membrane and that the</p><p>other enzyme is lost during isolation. The material to follow refers, therefore,</p><p>to the piericidin-sensitive enzyme as it occurs in purified preparations and in</p><p>ETP isolated directly from cells with the aid of Nossal or Braun shakers, or</p><p>DETERMINATION OF THE ACTIVITY OF DEHYDROGENASES 167</p><p>indirectly from mitochondria prepared with glusulase and then subjected to</p><p>sonication.</p><p>The enzyme resembles in many respects its counterpart in animal tissues,</p><p>including a high reactivity with ferricyanide and small or trivial reaction</p><p>with several other oxidants tested (89). The activity of NADH oxidase in</p><p>submitochondrial particles is measured by the method of Section IV.2, but</p><p>at 25” with 0.1 to 0.2 mg/ml added cytochrome c. The NADH-ferricyanide</p><p>assays are carried out as described in Section IV.4, but 3.3mM azide is</p><p>included in the case of particulate preparations. Measurement of V,,, is</p><p>essential, since competition between NADH and ferricyanide is evident even</p><p>at extremely low NADH concentrations (89).</p><p>The comments and method outlined above refer to NADH dehydrogenase</p><p>from the inner membrane of C. utilis cells in the late stationary phase. In the</p><p>log phase the cells contain an entirely different NADH dehydrogenase in the</p><p>inner membrane, with different catalytic properties and Fe-S signals. While</p><p>NADH dehydrogenase from log phase cells may also be assayed by the</p><p>ferricyanide method, the kinetics in this case are biphasic and it is not</p><p>known whether this assay measures the full NADH dehydrogenase activity</p><p>of the log phase enzyme (91a,b).</p><p>8. Determination of NADH Dehydrogenase Content by</p><p>Piericidin Binding</p><p>It is sometimes desirable to determine the NADH dehydrogenase content</p><p>of particles by a method independent of catalytic activity. It has been</p><p>established (6) that NADH dehydrogenase in membrane preparations binds</p><p>2 moles of piericidin A/mole of enzyme in extremely tight linkage (5). This</p><p>fact provides a chemical basis for the determination of their NADH de-</p><p>hydrogenase content.</p><p>Piericidin A is also bound at many other sites in mitochondria (the</p><p>so-called “unspecific binding sites”), but washing with 2% (w/v) bovine</p><p>serum albumin in 0.25M sucrose removes piericidin from these sites, leaving</p><p>the inhibitor bound only at NADH dehydrogenase (the “specific binding</p><p>sites”). In practice radiochemically pure piericidin A is incubated with</p><p>particles, unspecifically bound inhibitor is removed by washing the suspen-</p><p>sion twice by centrifugation with 2% bovine serum albumin in sucrose, and</p><p>residual radioactivity is counted. Since the specific activity of the 14C-</p><p>piericidin A used is known, a good approximation of the NADH de-</p><p>hydrogenase content may be calculated (5).</p><p>Preparations which have been exposed to bile salts or other detergents</p><p>yield a low piericidin titer by this method, presumably because the specific</p><p>binding sites involve lipids as well as the protein, and the former are</p><p>perturbed or removed by detergents (5). For the same reason the method is</p><p>168 THOMAS P. SINGER</p><p>not applicable to the soluble enzyme, which is lipid free. Although ETP</p><p>preparations from C. utilis are completely inhibited by piericidin A,</p><p>radiochemical determination of the NADH dehydrogenase content is ex-</p><p>tremely difficult in this case because bovine serum albumin partially dis-</p><p>sociates the inhibitor from the specific binding sites (92).</p><p>V. ASSAY OF CHOLINE DEHYDROGENASE</p><p>A. PRINCIPLE</p><p>The oxidation of choline via the complete respiratory chain (“choline</p><p>oxidase”) in rat liver mitochondria, the usual source of the enzyme, is</p><p>limited by the rate of entry of choline. This limitation may be overcome by</p><p>swelling agents, or by the inclusion of Ca2+ (0.25 to 0.75mM), although</p><p>there appears to be no agreement on the mechanism of stimulation by Ca2+.</p><p>Once provision is made to permit free entry of choline, the oxidation may be</p><p>readily measured polarographically, but semicarbazide must be included to</p><p>prevent further oxidation of betaine aldehyde, the product of choline oxida-</p><p>tion (93).</p><p>The most convenient reagent for assaying choline dehydrogenase activity</p><p>in appropriately treated mitochondria, in submitochondrial particles, and in</p><p>soluble preparations is phenazine methosulfate (PMS). In fact, in soluble</p><p>preparations it is the only known oxidant which functions satisfactorily (93).</p><p>In the original description of the PMS assay a manometric method was</p><p>used (93), which suffers from the limitations imposed by the rate of the</p><p>0,-leucophenazine methosulfate reaction (16). A later adaptation to the</p><p>Clark oxygen electrode (94) is even more subject to this source of error, since</p><p>in polarographic measurements the dissolved 0, concentration declines</p><p>constantly and thus the rate of reoxidation of the dye becomes increasingly</p><p>rate limiting in the course of the assay.</p><p>The procedure detailed below overcomes this limitation. It is an adapta-</p><p>tion of the spectrophotometric PMS-DCIP procedure (Section 11.4) to the</p><p>choline dehydrogenase system,</p><p>B. METHOD</p><p>REAGENTS. Phosphate buffer, 0.3M, pH 7.6.</p><p>Choline chloride, 0.5M.</p><p>KCN, 0.01M, neutralized, prepared fresh every 2 days and</p><p>DCIP, 0.05% (w/v) in O.OlM phosphate, pH 7.6, prepared</p><p>PMS, 0.33% (w/v) in water; cf. Section 11.4.</p><p>preserved at 0”.</p><p>by the method of Section 11.4.</p><p>DETERMINATION OF THE ACTIVITY OF DEHYDROGENASES 169</p><p>Procedure. Each of a series of six spectrophotometer cuvettes receives 0.5</p><p>ml of phosphate buffer, water to give a final volume of 3 ml during assay,</p><p>enzyme solution, and 0.3 ml of cyanide. After 3-min equilibration in a water</p><p>bath at 30°, the cuvettes are transferred to the thermostated (at 30") cell</p><p>compartment of a recording spectrophotometer, and 0.1 ml of DCIP solution</p><p>and varying volumes of PMS (0.33, 0.20, 0.15, 0.12, 0.10, 0.08, and 0.05 ml)</p><p>are added rapidly. The reaction is initiated by the addition of 0.1 ml of</p><p>choline, stirring quickly after the addition both of dyes and of choline. The</p><p>absorbance decline at 600 nm is recorded. The amount of enzyme used is</p><p>such as to give a 25 to 50% decrease in absorbance within 15 to 30 sec at a</p><p>0.25 or 0.5 absorbance unit setting on the recorder at the highest dye</p><p>concentration. Reciprocal decrease in absorbance per minute is plotted</p><p>against reciprocal volume of PMS and from the V,, (PMS) the amount of</p><p>choline oxidized is calculated, using the molar extinction coefficient of DCIP</p><p>at 600 nm, 19.1 X lo3. In a separate series of cuvettes the blank rate (i.e., no</p><p>choline) is determined and subtracted; as a rule, this is negligible with</p><p>soluble preparations of choline dehydrogenase.</p><p>The type of instrument recommended is described in Section IV.4. As in</p><p>the analogous succinate-PMS assay (Section 11.4), it is desirable to use</p><p>relatively high chart speeds (3 to 12 in./min) and to compensate for low</p><p>activity by increasing the expansion of the absorbance scale.</p><p>A typical assay is illustrated in Figure 16.</p><p>+ 3 m M AMYTAL</p><p>0</p><p>0</p><p>1 I I I 1 1 I 1</p><p>2 4 6 8 10 12 14 I6</p><p>I/ML PMS</p><p>Figure 16. Spectrophotometric assay of choline dehydrogenase. Experimental conditions were as</p><p>described in text, using 0.2 mg of a soluble enzyme, extracted from rat liver mitochdndria with</p><p>phospholipase A. From Hauber and Singer (95).</p><p>170 THOMAS P. SINGER</p><p>C. COMMENTS</p><p>In application of the method to mitochondria, in order to assure full</p><p>penetration of both choline and PMS, it is desirable to preincubate the</p><p>mitochondria with 1 pg purified phospholipase A (39)/mg protein for 5 to</p><p>10 min at room temperature. In view of the fact that initial rates are being</p><p>measured, further oxidation of betaine aldehyde is unlikely; hence the</p><p>addition of semicarbazide as a trapping agent is unnecessary.</p><p>As seen in Figure 16, the choline-PMS reaction is somewhat inhibited by</p><p>amytal. Although inhibition of choline oxidase by amytal has been known</p><p>for many years (96), the dehydrogenase itself appeared to be insensitive to</p><p>amytal in manometric assays (93). In later work, using a polarographic assay</p><p>but fixed PMS concentration (94), very extensive inhibition was reported. In</p><p>the more reliable spectrophotometric procedure the inhibition is trivial at</p><p>1mM concentration, is about 25% at 3mM concentration, and becomes</p><p>extensive only at lOmM concentration (95). This type of inhibition is not</p><p>related to the inhibition of NADH oxidase by barbiturates, for choline</p><p>oxidase and dehydrogenase are insensitive to rotenone and piericidin. A (95),</p><p>inhibitors which act at the same site as amytal in the NADH oxidase system</p><p>but are more powerful (97).</p><p>VI. ASSAY OF MITOCHONDRIAL</p><p>a-GLYCEROPHOSPHATE DEHYDROGENASE</p><p>A. PRINCIPLE</p><p>As is true of choline and succinate dehydrogenases, mitochondria1 a-</p><p>glycerophosphate dehydrogenase reacts most rapidly with PMS among</p><p>artificial electron acceptors tested; but, unlike the other enzymes, this</p><p>dehydrogenase retains considerable activity toward other dyes on solubiliza-</p><p>tion and purification (98,991. As a result, numerous assay methods have</p><p>been published, none of which is entirely satisfactory. A spectrophotometric</p><p>DCIP reduction procedure at fixed dye concentration (100) measures a</p><p>small and uncertain fraction of the activity because the slope in double</p><p>reciprocal plots of the DCIP assay is extreme (Figure 17); the same proce-</p><p>dure, using varying dye concentrations (99), is inconvenient for the same</p><p>reason; and the reduction of long-chain CoQ homologs gives a low rate and</p><p>shares the disadvantages detailed under the NADH-CoQ reductase assay</p><p>(Section IV.3). The manometric PMS procedure (98) and its polarographic</p><p>modification (101) are both subject to error because the rate of reoxidation</p><p>of the reduced dye by dissolved 0, may become rate limiting ( 1 6).</p><p>A recent spectrophotometric adaptation of the PMS method (102) is free</p><p>DETERMINATION OF THE ACTIVITY OF DEHYDROGENASES 171</p><p>0 1.0 2.0 3.0 4.0 PMS-DCIP</p><p>0 10 20 30 40 DCIPALONE</p><p>l/mM DYE</p><p>Figure 17. Comparison of DCIP and PMS-DCIP assays of soluble a-glycerophosphate de-</p><p>hydrogenase from pig brain mitochondria. From Salach and Bednarz (102).</p><p>from these shortcomings and appears to measure the full activity of the</p><p>dehydrogenase in mitochondria, submitochondrial preparations, and soluble</p><p>ones. As in the succinate dehydrogenase assay (Section 11.4), DCIP is used to</p><p>reoxidize reduced PMS and the absorbance decline at 600 nm is monitored.</p><p>As documented in Figure 17, the fact that DCIP itself is directly reduced by</p><p>the enzyme does not interfere, since the direct reduction of DCIP at all</p><p>actual dye concentrations is much slower than the PMS-mediated reduction.</p><p>At VmaX the PMS-DCIP and direct DCIP assays measure the same activity,</p><p>but the former is obviously more convenient because of a milder slope in</p><p>double reciprocal plots. In fact, except for the most accurate activity</p><p>determinations and for kinetic studies, one may use a fixed PMS concentra-</p><p>tion (the highest one specified below) without incurring significant error.</p><p>In studies dealing with reaction mechanisms of the dyes in which the dual</p><p>mechanisms of DCIP reduction may complicate interpretations, heart</p><p>muscle cytochrome c (0.02mM) may be substituted for DCIP as the terminal</p><p>electron acceptor, monitoring the increased absorbance at 550 nm (102),</p><p>since cytochrome c is not reduced directly by the enzyme in soluble prepara-</p><p>tions. This variation gives the same activity at V,,, (PMS) as does the</p><p>PMS-DCIP assay (102).</p><p>172 THOMAS P. SINGER</p><p>B. METHOD</p><p>REAGENTS. Phosphate buffer, 0.2M, pH 7.6, at 30".</p><p>DL-a-Glycerophosphate, 0.32M.</p><p>KCN, 0. IM, unneutralized.</p><p>DCIP, 0.05% (w/v) in 0.05M phosphate, pH 7.6 (protected</p><p>PMS, 0.33% (w/v), protected from light.</p><p>from light; see Section 11.4).</p><p>Procedure. In a series of six spectrophotometer cuvettes of 1 -cm light path</p><p>are placed, in the order given, 0.75 ml of buffer, water to give a 3-ml final</p><p>volume during assay, 0.3 ml of a-glycerophosphate, enzyme, and 0.025 ml of</p><p>cyanide. The cuvettes are covered, incubated for 3 min in a water bath at</p><p>30°, and transferred, one at a time, to the thermostated (at 30") cell</p><p>compartment of a recording spectrophotometer. In rapid succession 0.1 ml of</p><p>DCIP and 0.1 ml of PMS solution are added. The volumes of PMS used are</p><p>0.3, 0.2, 0.125, 0.1, 0.07, 0.05, and 0.03 ml. The cuvette contents are rapidly</p><p>mixed after the addition of PMS, and the absorbance decrease at 600 nm is</p><p>recorded. The chart speed recommended is 12 in./min, and the scale</p><p>expansion 0.5 absorbance unit full scale. The amount of enzyme used is such</p><p>as to cause 25 to 50% decrease in absorbance in the initial 30 sec at this scale</p><p>expansion.</p><p>The results are calculated from double reciprocal plots of decrease in</p><p>absorbance against PMS concentration, and the resulting V,, value is</p><p>converted to concentration by the use of the molar extinction coefficient of</p><p>DCIP at 600 nm (19.1 X lo3).</p><p>The equipment recommended is the same as in the method of Section</p><p>IV.4.</p><p>Acknowledgment</p><p>The original studies reported here were aided by grants from the National</p><p>Institutes of Health (HE 10027), the National Science Foundation (GB</p><p>20814), and the American Cancer Society (BC 46 A).</p><p>References</p><p>1.</p><p>2.</p><p>3.</p><p>4.</p><p>5 .</p><p>T. P. Singer and E. B. Kearney, in Methodr of Biochemical Analysrr, Vol. 4, D. Click, Ed.,</p><p>Interscience, New York, 1957, p. 307.</p><p>T. P. Singer, in Comprehmrivc Biochemistry, Vol. 14, M. Florkin and E. H. Stotz, Eds.,</p><p>Elseivier, Amsterdam, 1966, p. 127.</p><p>T. P. Singer, in The Enzymes, Vol. VII, P. Boyer, H. A. Lardy, and K. Myrback, Eds.,</p><p>Academic Press, New York, 1963, p. 345.</p><p>T. P. Singer, J. Salach, P. Hemmerich, and A. Ehrenberg, in Methods in Enzymolosy, Vol.</p><p>18B, D. McCormick and L. Wright, Eds., Academic Press, New York, 1971, p. 416.</p><p>D. J. Horgan, H. Ohno, T. P. Singer, and J. E. Casida,J. Bid. Chem., 243, 5967 (1968).</p><p>DETERMINATION OF THE ACTIVITY OF DEHYDROGENASES 173</p><p>6.</p><p>7.</p><p>8.</p><p>9.</p><p>10.</p><p>M. Gutman, T. P. Singer, and J. E. Casida, J. Biol. Chem., 245, 1992 (1970).</p><p>V. Massey and T. P. Singer, J. Bid. Chn. , 228, 263 (1957).</p><p>T. P. Singer, in Molecular Euolution, Vol. 2, E. Schoffeniels, Ed., North Holland, Amster-</p><p>dam, 1971, p. 203.</p><p>T. E. King, Biochim. Biophys. Acta, 47, 430 (1961).</p><p>T. P. Singer, M. Gutman, and E. B. Kearney, in Biochemistry and Biophysics of Mitochondria1</p><p>Membranes, G. F. Azzone, E. Carafoli, A. L. Lehninger, E. Quagliariello, and N.</p><p>Siliprandi, Eds., Academic Press, New York, 1972, p. 41.</p><p>11. K. A. Davis and Y. Hatefi, Biochmistty, 10, 2509 (1971).</p><p>12. C. J. Coles, H. Tisdale, W. C. Kenney, and T. P. Singer,J Biol. Chn. , 249, 381 (1974).</p><p>13. T. P. Singer, E. B. Kearney, and W. C. Kenney, Adu. Enzymol., 36, 189 (1972).</p><p>14. T. P. Singer and C. J. Lusty, Biochem. Biophys. Res. Commun., 2, 276 (1960).</p><p>15. M. Klingenberg, Eur. J. Biochn., 13, 247 (1970).</p><p>16. 0. Arrigoni and T. P. Singer, Nature, 193, 1256 (1962).</p><p>17. T. P. Singer, G. Oestreicher, P. Hogue, J. Contreiras, and I. Brandao, Plant Physzol., 52,</p><p>616 (1973).</p><p>18. A. Giuditta and T. P. Singer, J. Bid. Chon., 234, 666 (1959).</p><p>19. E. Rossi, B. Norling, B. Person, and L. Emster, Eur J. Biochem., 16, 508 (1970).</p><p>20. W. P. Zeylemaker, A. D. M. Klaase, E. C. Slater, and C. Veeger, Biochim. Biophys. Acta,</p><p>198,415 (1970).</p><p>21. W. P. Zeijlemaker, D. V. Dervartanian, C. Veeger, and E. C. Slater, Biochim. Biopbs.</p><p>Ach, 178, 213 (1969).</p><p>22. W. C. Hanstein, K. A. Davis, M. A. Ghalambor, and Y. Hatefi, Biochemistry, 10, 2517</p><p>(1971).</p><p>23. E. B. Kearney and T. P. Singer, J. Bid. Chm., 219, 963 (1956).</p><p>24. V. Massey and T. P. Singer, J. Bid. C h n . , 229, 755 (1957).</p><p>25. E. B. Kearney, J. Biol. Chem., 229, 363 (1957).</p><p>26. T. Kimura, J. Hauber, and T. P. Singer,J. Bid. C h n . , 242, 4987 (1967).</p><p>27. M. Gutman, E. B. Kearney, and T. P. Singer, Biochemistry, 10, 2726 (1971).</p><p>28. M. Gutman, E. B. Kearney, and T. P. Singer, Biochemistry, 10, 4763 (1971).</p><p>29. E. B. Kearney, M. Mayr, and T. P. Singer, Biochm. Biophys. Res. Commun., 46,531 (1972).</p><p>30. E. B. Kearney, B. Ackrell, and M. Mayr, Biochm. Biophys. Rcs. Commun., 49, 1115 (1972);</p><p>J. Bioi. Chem. (in press).</p><p>31. T. P. Singer, E. B. Kearney, and M. Gutman, in Biochemical Regulatory Mechanisms in</p><p>Eukaryotic Ceh, E. Kun and S. Grisolia, Eds., Wiley-Interscience, New York, 1972, p. 271.</p><p>32. M. B. Thorn, Biochem. J., 85, 116 (1962).</p><p>33. D. Dervartanian and C. Veeger, Biochim. Biophys. Ach, 105, 424 (1965).</p><p>34. W. H. Walker, T. P. Singer, S. Ghisla, P. Hemmerich, U. Hartmann, and E. Zwotek,</p><p>Eur. J. Biochem., 26, 279 (1972).</p><p>35. T. P. Singer, J. Hauber, and E. B. Kearney, Biochem. Biqhys. Res. Commun., 9, 146 (1962).</p><p>36. T. P. Singer and T. Cremona, in Oxygen in the Animal Organism, F . Dickens and E. Neil,</p><p>Eds., Pergamon Press, London, 1964, p. 179.</p><p>37. P. Bernath and T. P. Singer, in Methods in Enzynolou, Vol. V. S. Colowick and N. 0.</p><p>KapJan, Eds., Academic Press, New York, 1962, p. 597.</p><p>38. D. Ziegler and J. S. Rieske, in Methodr in Enzymolou, Vol. X, R. W. Estabrook and M.</p><p>Pullman, Eds., Academic Press, New York, 1967, p. 231.</p><p>39. T. Cremona and E. B. Kearney, J. Bid. Chem., 239, 2328 (1964).</p><p>40. A. Giuditta and T. P. Singer, J. Bid. Chem., 234, 662 (1959).</p><p>41. T. E. King, J. Bid. C h n . , 238, 4032, 4037 (1963).</p><p>42. C. Veeger, D. Dervartanian, and W. P. Zeylemaker, in Methds in Enrymology, Vol. XIII,</p><p>J. M. Lowensten, Ed., Academic Press, New York, 1969, p. 81.</p><p>174 THOMAS P. SINGER</p><p>43. D. Keilin and T. E. King, Proc. Roy. Soc. (London), B152, 163 (1960).</p><p>44. T. Kimura, J. Hauber, and T. P. Singer, Nature, 198, 362 (1963).</p><p>45. M. L. Baginsky and Y. Hatefi,J. Biol. C h . , 244, 5313 (1969).</p><p>46. T. Kimura and J. Hauber, Biochem. Biophys. Res. Commun., 13, 169 (1963).</p><p>47. T. E. King, in Melhadc in E~ymologv, Vol. X, R. W. Estabrook and M. Pullman, Eds.,</p><p>Academic Press, New York, 1967, p. 202.</p><p>48. T. P. Singer and E. B. Kearney, in Vitamin Mefubolism, W. Umbreit and H. Molitor, Eds.,</p><p>Pergamon Press, London, 1959, p. 209.</p><p>49. E. B. Kearney, J. Biol. C h . , 235, 865 (1960).</p><p>50. D. R. Patek and W. R. Frisell, Arch. Biocb . Biophys., 150, 339 (1972).</p><p>51. D. R. Patek and W. R. Frisell, Arch. Biochcm. Biophys., 150, 347 (1972).</p><p>52. W. H. Walker, and T. P. Singer,J. Biol. Chem., 245, 4224 (1970).</p><p>53. W. C. Kenney, W. H. Walker, E. B. Kearney, R. Seng. T. P. Singer, J. R. Cronin, and</p><p>R. Hendricks, Z Naturforsch., 276, 1069 (1972).</p><p>54. E. B. Kearney, J. I. Salach, W. H. Walker, R. Seng, W. C. Kenney, E. Zeszotek, and T.</p><p>P. Singer, EurJ. Biochem., 24, 321 (1971).</p><p>55. W. H. Walker, E. B. Kearney, R. Seng, and T. P. Singer, Eur.J. Biochnn., 24,328 (1971).</p><p>55a. W. C. Kenney, D. E. Edmown, T. P. Singer, Biochon. Biophysics Rcs. Commun. 57, 106</p><p>55b. W. C. Kenney, D. E. Edrnonson, and T. P. Singer, J. C. Schabort, D. J. Steenkemp,</p><p>FEES Letts. (article in press).</p><p>56. T. P. Singer, E. Rocca, and E. B. Kearney, in Fiavins and Fiauoproieinr, E. C. Slater, Ed.,</p><p>Elsevier, Amsterdam, 1966, p. 391.</p><p>57. B. Mackler, P. J. Collier, H. M. Duncan, N. A. Rao, and F. M. Huennekens, J. Biol.</p><p>Chon., 237, 2968 (1962).</p><p>58. H. R. Mahler, B. Mackler, S. Grandchamp, and P. P. Slonimski, Biochemistry, 3, 668</p><p>59. J. Hauber and T. P. Singer, Eur.J. Biochmt., 3, 107 (1967).</p><p>60. M. G. P. J. Warringa, 0. H. Smith, A. Giuditta, and T. P. Singer,J. Biol. Chem., 230, 97</p><p>(1958).</p><p>61. C. A. Hirsch, M. Rasminsky, B. D. Davis, and E. C. C. Lin, J. Biol. Chm., 238, 3770</p><p>(1963).</p><p>62. M. G. P. J. Warringa and A. Giuditta,J. Biol. Chon., 230, 111 (1958).</p><p>63. F. J. S. Lara, Biochim. Biophy. Ada, 33, 565 (1959).</p><p>64. G. Oestreicher, P. Hogue, T. P. Singer, J. Contreiras, and I. Brandao, Plant Physiol., 52,</p><p>613, 622 (1973).</p><p>65. T. P. Singer and E. B. Kearney,J. Biol. Chon., 183, 409 (1950).</p><p>66. J. 0. D. Coleman, and J. M. Palmer, EurJ. Biochem., 26, 499 (1972).</p><p>67. M. A. Matlib, R. C. Kirkwood, and J. E. Smith,J. Exp. Bot., 22, 291 (1971).</p><p>68. T. P. Singer and M. Gutman, Adv. Enrymoi., 34, 79 (1971).</p><p>69. H. Watari, E. B. Kearney, and T. P. Singer,J. Bid. Chon ., 238, 4063 (1963).</p><p>70. T. Cremona, E. B. Kearney, M. Villavicencio, and T. P. Singer, Biochm. Z., 338, 407</p><p>71. J. M. Machinist and T. P. Singer, Proc. Nal. Acad. Sci. U.S., 53, 467 (1965).</p><p>72. M. Gutman, T. P. Singer, H. Beinert, and J. E. Casida, PTOC. Mat. Acad. Sci. U.S., 65, 763</p><p>(1970).</p><p>73. M. Gutman, T. P. Singer, and H. Beinert, Biochemistty, 11, 556 (1972).</p><p>74. R. L. Ringler, S. Minakami, and T. P. Singer,J. Bid. Chem., 238, 801 (1963).</p><p>75. J. Salach, T. P. Singer, and P.</p><p>Bader,J. Bid. Chem., 242, 4555 (1967).</p><p>76. R. L. Pharo, L. A. Sordahl, S. R. Vyas, and D. R. Sanadi, J. Bid. Chem., 241, 4771</p><p>(1966).</p><p>(1974).</p><p>( 1964).</p><p>( 1963).</p><p>DETERMINATION OF THE ACTIVITY OF DEHYDROGENASES 175</p><p>77. H. R. Mahler, N. K. Sarkar, L. P. Vernon, and A. R. Alberty, J. Biol. Chem., 199, 585</p><p>(1952).</p><p>78. D. R. Biggs, J. Hauber, and T. P. Singer, J. Biol. Chem., 242, 4563 (1967).</p><p>79. H. Beinert, G. Palmer, T. Cremona, and T. P. Singer, J. Biol. Chem., 240, 475 (1965).</p><p>80. G. Oestreicher, P. Hogue, and T. P. Singer, article to be published.</p><p>81. C. J. Coles, unpublished data.</p><p>82. S. Minakami, R. L. Ringler, and T. P. Singer, J. Biol. Chem., 237, 569 (1962).</p><p>83. T. Cremona and E. B. Kearney, J. Bid. Chem., 240, 3645 (1965).</p><p>84. S. Minakami, T. Cremona, R. L. Ringler, and T. P. Singer, J. Biol. C h n . , 238, 1529</p><p>(1963).</p><p>85. T. P. Singer, in Flauins and Flauoproteins, E. C . Slater, Ed., Elsevier, Amsterdam, 1966, p.</p><p>489.</p><p>86. N. A. Rao, S. P. Felton, F. M. Huennekens, and B. Mackler, J. Biol. Chem., 238, 449</p><p>(1963).</p><p>87. T. P. Singer and E. B. Kearney in Symposium on Redox Functions of Cytoplasmic Structures, Th.</p><p>Bucher, Ed., Vienna, 1962, Preprints, p. 251.</p><p>88. A. M. Stein, N. 0. Kaplan, and M. M. Ciotti, J. Biul. Chem., 234, 979 (1959).</p><p>89. D. R. Biggs, H. Nakamura, E. B. Kearney, E. Rocca, and T. P. Singer, Arch. Biochem.</p><p>Biophys., 137, 12 (1970).</p><p>90. T. Ohnishi, in Flauins and Flauoproteins, H. Kamin, Ed., University Park Press, Baltimore,</p><p>1971, p. 691.</p><p>91. S. 0. C. Tottmar and C. I. Ragan, Biochem J., 124, 853 (1971).</p><p>91a. C. J. Cobley, S. Grossman, H. Beinert, and T. P. Singer, Biochem. Biophys. Res. Commun.,</p><p>53, 1273 (1973).</p><p>91b. S. Grossman, C. J. Cobley, T. P. Singer, and H. Beinert, J. Biol. Chem. (article in press).</p><p>92. C. J. Coles, M. Gutman, and T. P. Singer,J. Bid. Chem. (article in press).</p><p>93. G. Rendina and T. P. Singer, J. Bid. Chem., 234, 1605 (1959).</p><p>94. D. D. Tyler, J. Gonze, and R. W. Estabrwk, Arch. Biochem. Biophys., 115, 373 (1966).</p><p>95. J. Hauber and T. P. Singer, unpublished data.</p><p>96. L. Ernster, 0. Jalling, H. E w , and 0. Lindberg, Exp. Cell Res., 3, Suppl. 124 (1955).</p><p>97. D. J. Horgan, T. P. Singer, and J. E. Casida, J. Biol. Chem., 243, 834 (1968).</p><p>98. R. L. Ringler and T. P. Singer, J. Biol. Chem., 234, 2211 (1959).</p><p>99. A. P. Dawson and C. J. R. Thorne, Biochem. J., 114, 35 (1969).</p><p>100. K. H. Ling, S. H. Wu, S. M. Ting, and T. C. Tung, Proceedings of the International</p><p>Symposium on Enzyme Chemkty, Tokyo and Kyoto, Maruzen, Tokyo, 1958, p. 268.</p><p>101. A. P. Dawson and C. J. R. Thorne, Biochm. J., 111, 27 (1969).</p><p>102. J. I. Salach and J. Bednan, Arch. Biochmz. Biophys., 157, 133 (1973).</p><p>reactants</p><p>in intact mitochondria, and multiple reaction sites for electron acceptors. In</p><p>certain tissues (e.g., heart), which contain only one type of covalently bound</p><p>flavin, the fluorometric determination of histidyl-flavin offers an unam-</p><p>biguous method for determing the concentration of succinate dehydrogenase</p><p>(4). This, in turn, serves as a basis for calculating the turnover number of the</p><p>enzyme (i.e., catalytic center activity) regardless of the purity of the prepara-</p><p>tion. Determination of the “specifically bound” piericiden A titer (5 ,6) offers</p><p>an analogous chemical method for determining the concentration of NADH</p><p>dehydrogenase in particulate preparations of varying complexity from mi-</p><p>tochondria to Complex I, but the procedure is not applicable to soluble</p><p>preparations, since the lipids involved in the piericidin binding sites (5) are</p><p>removed during extraction. Selective chemical methods for measuring the</p><p>126 THOMAS P. SINGER</p><p>concentrations of choline and a-glycerophosphate dehydrogenases are not</p><p>yet available.</p><p>11. ASSAY OF SUCCINATE DEHYDROGENASE AND</p><p>SUCCINOXIDASE IN ANIMAL TISSUES</p><p>1. General Considerations</p><p>Succinate dehydrogenase is reversible in aerobic cells, and thus, in prin-</p><p>ciple, the enzyme may be assayed in either direction of catalysis (7). The</p><p>aerobic type of enzyme, however, reacts much faster in the direction of</p><p>succinate oxidation than of fumarate reduction (8). Hence assays based on</p><p>the forward reaction are generally more sensitive.</p><p>Depending on the experimental material (mitochondria, inner</p><p>mitochondria1 membranes, membrane fragments, or soluble enzyme), a</p><p>variety of electron acceptors may be used to follow the rate of succinate</p><p>oxidation, but only a few of these are capable of measuring the full activity</p><p>of the dehydrogenase. One reason for this is that the turnover number of the</p><p>dehydrogenase in mammalian systems is much higher than the rate of</p><p>electron transport through the respiratory chain, so that succinoxidase and</p><p>succinate-cytochrome c reductase activities in membrane preparations are</p><p>always lower than succinate dehydrogenase activity. Furthermore, externally</p><p>added oxidants, such as cytochrome c, usually do not react rapidly with</p><p>intact membrane preparations, unless some treatment is applied to “open”</p><p>the system. No such impedence exists, of course, in the reaction of 0, with</p><p>the cytochrome oxidase end of the system.</p><p>Succinate dehydrogenase assays involve the use of oxidants such as ferri-</p><p>cyanide or N-alkylphenazonium salts. Certain authors have found the use of</p><p>artificial electron acceptors distasteful on the grounds that they do not reflect</p><p>certain “native properties” of the enzyme, such as the ability to recombine</p><p>with the respiratory chain (9). It must be understood that the assays to be</p><p>described are designed to measure the catalytic activity of the enzyme and</p><p>thus do not necessarily bear any relation to regulatory properties or</p><p>“reconstitutive” properties (see Section 11.7), which are expressions of pro-</p><p>tein conformation not necessarily involving the catalytic site. Nevertheless,</p><p>the measurement of succinate dehydrogenase activity with phenazine</p><p>methosulfate in mitochondria accurately reflects changes in the state of</p><p>activation of the enzyme in metabolic transitions, which may be directly</p><p>correlated with substrate flux (10). Moreover, a purified succinate de-</p><p>hydrogenase preparation with normal catalytic and “reconstitutive” proper-</p><p>ties but modified regulatory properties (1 2,13) has been described (1 1). Thus</p><p>it must be emphasized that the assay selected should reflect the property of</p><p>DETERMINATION OF THE ACTIVITY OF DEHYDROGENASES 127</p><p>the enzyme to be investigated: catalytic activity, regulatory properties, or</p><p>" reconstitutive" properties.</p><p>The use of artificial electron acceptors in the assay of the dehydrogenase</p><p>in complex preparations (whole cells, mitochondria) involves two problems:</p><p>permeability and multiple reaction sites. Neither phenazine methosulfate</p><p>(14) nor ferricyanide (15) can freely penetrate the inner mitochondrial</p><p>membrane, so that in assaying succinate dehydrogenase activity in mam-</p><p>malian mitochondria with these dyes prior treatment with phospholipase A,</p><p>Ca2+, or both (16) is required to assure free penetration of the dye. In the</p><p>case of higher plants neither of these agents seems to overcome the permea-</p><p>bility barrier, so that at present the true activity of the enzyme can be</p><p>measured only after sonic disruption of the mitochondria (1 7). When tissues</p><p>other than those which have been tested (heart, liver, brain, aerobic yeast)</p><p>are examined, procedures which will assure free penetration of the oxidant</p><p>must be ascertained. If the succinate-PMS activity of a mitochondria1</p><p>preparation, assayed after full activation, increases on sonication or freeze-</p><p>thawing, penetration of the dye is probably rate limiting. The problem does</p><p>not arise with inner membrane preparations (ETP, ETP,, ESP), since these</p><p>are inverted, so that the dehydrogenase, originally located on the inside of</p><p>the inner membrane, is on the outside in the derived vesicles.</p><p>The second problem involved in the use of artificial electron acceptors</p><p>concerns the fact that both PMS and ferricyanide react a t more than one</p><p>site in the respiratory chain. Many years ago it was proposed that PMS has</p><p>two reaction sites at or near the dehydrogenase, both being required for full</p><p>catalytic activity, one of which is lost on extraction of the enzyme or on</p><p>treatment with cyanide (18). This proposal was based on the observation</p><p>that treatment of the membrane-bound form of the dehydrogenase with</p><p>cyanide or extraction in soluble form resulted in 50 to 70% lower turnover</p><p>number in the succinate-PMS assay, without a corresponding loss in the</p><p>fumarate-FMNH, activity of the enzyme. The idea was revived by Rossi et</p><p>al. (19) in recent studies showing that pentane extraction of CoQ,, results in</p><p>some 50% decline of succinate-PMS activity, but that full activity is regained</p><p>on reincorporation of CoQ,, into the extracted particles. One of the two</p><p>reaction sites of PMS appears to be on the dehydrogenase itself, while the</p><p>second site may be CoQ,,H, (13).</p><p>Similar complications arise with ferricyanide as the oxidant. In addition</p><p>to reacting with the dehydrogenase itself, ferricyanide also accepts electrons</p><p>at the level of cytochrome c or c,. While the latter reaction is readily blocked</p><p>with antimycin A in preparations of the complete respiratory chain, it is not</p><p>certain that a single reaction site operates at the level of the dehydrogenase.</p><p>A practical reason for considering this point is the question of whether the</p><p>PMS-DCIP and ferricyanide assays can be used interchangeably, that is,</p><p>128 THOMAS P. SINGER</p><p>whether results based on these alternative procedures are comparable.</p><p>In our early studies, when the manometric ferricyanide assay (1) was used,</p><p>the turnover number of the enzyme in the ferricyanide assay was always</p><p>much lower than in the PMS assay (both based on V,,, for the dye), in</p><p>every type of enzyme preparation and mitochondrion studied. Later</p><p>Zeylemaker et al. (20) discovered that bicarbonate is a competitive inhibitor</p><p>of the enzyme, so that low activity with ferricyanide in the manometric assay</p><p>could have been due to the high bicarbonate concentration. In fact, it was</p><p>reported (21) that at 25" the spectrophotometric PMS and ferricyanide</p><p>assays yield the same activity. Comparable activities in the two assays have</p><p>also been noted in this laboratory at 25", but, more frequently, the activity</p><p>in the ferricyanide assay was only about 50% of that obtained with PMS,</p><p>using soluble preparations and excluding bicarbonate. Moreover, as the</p><p>temperature of the assay is increased, the deviation between the two assays</p><p>becomes more marked. In agreement with the report of Hanstein et al. (22)</p><p>and our early studies (23,24) we believe that, at least at 37 to 38", the</p><p>ferricyanide assay</p><p>does not measure the full activity of the enzyme and that</p><p>the deviations between the PMS and ferricyanide assays appear to be due to</p><p>anomalies in the reaction of ferricyanide with the enzyme. This point is</p><p>further discussed and documented in Section 11.5.</p><p>Regardless of the type of electron acceptor or preparation employed, an</p><p>absolute requirement for reliable assays of the enzyme is that it be fully</p><p>activated. Succinate dehydrogenase from all aerobic cells examined is acti-</p><p>vated by preincubation with substrates or competitive inhibitors (25). The</p><p>process is characterized by a high energy of activation, suggesting a confor-</p><p>mation change, and is reversible (26). More recently ITP, IDP, acid pH,</p><p>high concentrations of certain anions, and reduced CoQ,, have also been</p><p>shown to activate the enzyme; in mitochondria ATP and all metabolites</p><p>which reduce endogenous CoQ,, (a-glycerophosphate, choline, NAD-linked</p><p>substrates) also activate the enzyme (27-29). One mechanism of activation,</p><p>at least by certain agents, such as anions, CoQH,, and inosine nucleotides,</p><p>appears to involve the removal of very tightly bound oxalacetate from the</p><p>dehydrogenase, although rapid deactivation-activation of the oxalacetate-</p><p>free enzyme also occurs (30). For details of this phenomenon and its</p><p>regulatory significance the reader is referred to recent reviews (13,3 1).</p><p>What is important from the standpoint of this article is that provisions</p><p>must be made to fully activate the enzyme before assay; otherwise an</p><p>uncertain fraction of the activity, which may be as little at 1% of the total</p><p>(26), may be measured, and nonlinear kinetics may result. The need for this</p><p>precaution has been eloquently emphasized by Thorn (32) but has been</p><p>frequently ignored.</p><p>The type of activator to be used and the conditions for activation vary to</p><p>DETERMINATION OF THE ACTIVITY OF DEHYDROGENASES 129</p><p>some extent with the type of preparation. Succinate is satisfactory with</p><p>vertebrate and plant preparations, as well as those from aerobic</p><p>microorganisms, regardless of the state of purification of the enzyme. If the</p><p>design of the experiment is such that prior treatment with succinate is</p><p>undesirable, one may use malonate for activation, correcting for the inhibi-</p><p>tion by the malonate carried over into the assay; in intact mitochondria</p><p>ATP (1mM or less) is an excellent activator; in submitochondrial or soluble</p><p>preparations IDP or ITP (-20mM) or relatively high concentrations of</p><p>Br - or similar anions provide rapid and complete activation, particularly in</p><p>the presence of semicarbazide.</p><p>In our original paper (16) on the spectrophotometric PMS-DCIP assay an</p><p>additional precaution listed was the inclusion of cysteinesulfinate and tran-</p><p>saminase in the assay of the enzyme in membrane preparations containing</p><p>malate dehydrogenase and NAD. The purpose of this was to remove the</p><p>oxalacetate formed by malate dehydrogenase action, lest it inhibit succinate</p><p>dehydrogenase. This precaution is no longer recommended for the following</p><p>reasons. First, it has been found (33) that both D- and L-malate are oxidized</p><p>by succinate dehydrogenase itself. The oxidation is so rapid and such a low</p><p>concentration of oxalacetate is required to deactivate the enzyme that it is</p><p>doubtful whether the transaminase can effectively compete with the de-</p><p>hydrogenase for oxalacetate. On the other hand, if the succinate concentra-</p><p>tion is in the millimolar range, the oxidation by succinate dehydrogenase of</p><p>any L-malate arising from the fumarate by the combined action of fumarase</p><p>and succinate dehydrogenase is probably effectively prevented. Initial rate</p><p>measurements and maintenance of a relatively high succinate concentration</p><p>(20mM) in the assay are also probably the best available means for</p><p>preventing inhibition by oxalacetate arising from malate dehydrogenase</p><p>activity. When the succinate concentration is very low (</p><p>with either 40mM succinate or ImM malonate for 10</p><p>to 20 min at 30" (25,26). When malonate is used as the activator, the</p><p>process is carried out in test tubes and the protein concentration is adjusted</p><p>so that a 25- to 5O-pl aliquot of the activated enzyme will give a suitable rate</p><p>in the subsequent activity measurement. In this manner the inhibition by</p><p>the small amount of malonate carried over into the assay, in the presence of</p><p>20mM succinate, is very slight and may be readily corrected for by adding</p><p>an identical amount of malonate to a succinate-activated sample and</p><p>measuring the resulting inhibition. When succinate is used as the activator,</p><p>the particles are placed in the main compartment of a small Thunberg tube</p><p>at 0", and neutralized succinate in the side arm. In a typical experiment</p><p>0.45 ml particle suspension is placed in the main part of the tube and 0.05</p><p>ml of 0.4M succinate in the side arm. The tube is twice evacuated and filled</p><p>with N, or He. After 3 min temperature equilibration at 30" khe contents of</p><p>the side arm are tipped. After 15 min the tube is opened, and the activated</p><p>enzyme is used immediately as the last component added to start the assay.</p><p>The time required for full activation depends on the protein concentration</p><p>and the type of particle preparation. Activation is faster in mitochondria</p><p>than with submitochondrial particles and is inversely related to protein</p><p>concentration. In general, if the protein concentration during activation is</p><p>less than 5 mg/ml, 10 min at 30" may suffice; at and above 10 mg/ml a full</p><p>132 THOMAS P. SINGER</p><p>20 min may be required. The activation time must be determined for each</p><p>type of enzyme preparation.</p><p>Succinoxidase activity is determined polarographically with a Clark or a</p><p>vibrating Pt electrode in a closed chamber thermostated at the requisite</p><p>temperature (usually 30”). The reaction vessel (usually 3 ml) contains</p><p>80mM potassium phosphate-50pM EDTA, pH 7.5 to 7.6, 20mM succinate,</p><p>and, in the case of cytochrome-deficient preparations, 0.5 mg of cytochrome</p><p>c (Sigma, Type VI). With all components except the enzyme present and a t</p><p>temperature equilibrium, the activated particles are added in a small</p><p>volume (10 to 20 pl) to start the reaction. The sample size is adjusted so as to</p><p>use up at least 25% of the 0, present in a convenient time period (1 to 5</p><p>min).</p><p>3. Succinate-Coenzyme Q Reductase Assay</p><p>A. PRINCIPLE</p><p>The oxidation of succinate with an externally added C o Q derivative as</p><p>oxidant may be coupled to the reduction of DCIP or of ferricyanide (38).</p><p>The use of DCIP as terminal oxidant, in the presence of cyanide to block its</p><p>reoxidation, is more advantageous since DCIP is reduced only very slowly</p><p>without added CoQ, at least in preparations in which C o Q reductase</p><p>activity is commonly measured (e.g., Complex II), while ferricyanide is</p><p>directly reduced, so that the rate measured in the presence of added C o Q is</p><p>the sum of the direct and of the CoQ-mediated activities. The higher</p><p>extinction coefficient of DCIP than of ferricyanide also provides a more</p><p>sensitive method.</p><p>In soluble, purified preparations from animal tissues C o Q reductase</p><p>activity is very low, but in particulate preparations of the “open” type (i.e.,</p><p>those in which added C o Q reacts freely with the respiratory chain), such as</p><p>Complex 11, the rate of reduction of CoQ, and of Co,, by succinate equals</p><p>the succinate-PMS activity. The reaction probably proceeds by way of the</p><p>internal CoQ,,, which may serve as the immediate oxidant of the de-</p><p>hydrogenase and is strictly lipid dependent. With CoQ, and CoQ,, which</p><p>are water soluble, the addition of detergents does not seem necessary,</p><p>although it has been recommended (38). Unfortunately neither CoQ, nor</p><p>CoQ, is commercially available at this time. Optimal assay conditions for</p><p>the use of CoQs or CoQ,,, which are commercially available but are water</p><p>insoluble, have not been elaborated.</p><p>B. METHOD</p><p>The procedure to follow is designed for a 3-ml reaction volume but may</p><p>be readily scaled down, using suitable semimicrocuvettes. Succinate (60</p><p>DETERMINATION OF THE ACTIVITY OF DEHYDROGENASES 133</p><p>pmoles), phosphate buffer, pH 7.5 (180 pmoles), EDTA (0.15 pmoles),</p><p>enzyme, and neutral cyanide (prepared and preserved as described in</p><p>Section 11.4 on the PMS-DCIP procedure), 3 pmoles, are placed in a</p><p>covered spectrophotometer cuvette containing 2.8 ml total volume. For</p><p>activation the solution is incubated in a water bath for 5 min at 38" or 10</p><p>min at 30", depending on the assay temperature selected. The cuvette is</p><p>then transferred to the cell compartment of a thermostated recording</p><p>spectrophotometer, and the reaction is started by the addition of 0.1 ml each</p><p>of an aqueous solution of 0.75mM CoQ, or CoQ, and of 0.05% (w/v)</p><p>neutral DCIP or 0.03M ferricyanide. With DCIP the wavelength used is 600</p><p>nm and the millimolar extinction coefficient for calculations is 19.1; with</p><p>ferricyanide the wavelength is 420 nm and the coefficient is 1.0. The</p><p>spectrophotometer used should have an optical density offset and a rapidly</p><p>adjustable scale expansion varying from 0.1 to 1 .O absorbance unit full scale,</p><p>as in the Gilford 222 or 240 instruments. The recorder should have several</p><p>chart speeds in the range of 1 to 12 in./min which may be rapidly changed.</p><p>This combination permits the assay of samples of widely varying activities,</p><p>since change of the scale expansion (range) of the detecting instrument</p><p>and/or of the recorder speed is possible during the first few seconds after</p><p>initiation of the reaction, so as to yield a tracing with a slope in the 30 to 60"</p><p>range.</p><p>4. Phenazine Methosulfate Assay</p><p>A. PRINCIPLE</p><p>The assay is based on the reduction of PMS by succinate and its</p><p>dehydrogenase. Reduced PMS is immediately reoxidized by DCIP; bleach-</p><p>ing of the latter dye is estimated spectrophotometrically (16).</p><p>B. METHOD</p><p>REAGENTS. Potassium phosphate buffer, 0.3M, pH 7.5, at room tempera-</p><p>ture.</p><p>Sodium succinate, 0.2M, adjusted to pH 7.5 at room tem-</p><p>perature.</p><p>Potassium cyanide, 0.0 1 M , neutralized. One millimole of</p><p>KCN is dissolved in about 95 ml cold distilled water in a</p><p>glass-stoppered volumetric flask, and 0.85 ml cold lNHCl is</p><p>rapidly added. The flask is kept stoppered in ice for 5 to 10 min</p><p>to permit reabsorption of the HCN and is then diluted to 100</p><p>ml. Fresh solution is prepared every 2 days.</p><p>DCIP, 0.05% (w/v) in 0.01M potassium phosphate, p 7.5. A</p><p>30-fold dilution should have an absorbance between 1.05 and</p><p>134 THOMAS P. SINGER</p><p>1.25 in a 1-cm light path at 600 nm. The solution may be kept</p><p>for at least a week in the cold, protected from light. Note that</p><p>the purity of commercial DCIP is highly variable; in our</p><p>experience the product of General Biochemicals, Inc., appears</p><p>to be most satisfactory. Some batches of this company's product</p><p>dissolve with difficulty (or precipitate in the cold), however,</p><p>and need to be sonicated.</p><p>PMS, 0.33% (w/v) in water. Although the synthesis of PMS</p><p>is very simple ( l), the products marketed by several biochemi-</p><p>cal supply houses are contaminated to varying degrees with</p><p>decomposition products which inhibit the dehydrogenase. Dur-</p><p>ing the past few years the only uniformly satisfactory material</p><p>has been that supplied by the Sigma Chemical Co. Solutions of</p><p>PMS are perfectly stable if protected from light and stored at</p><p>The cuvettes are covered</p><p>immediately after the addition of KCN and incubated for 5 to 7 min at 38"</p><p>to permit full activation. (If the assay is to be conducted at 30", 8 to 15 min</p><p>may be required for full activation, depending on the protein concentration.)</p><p>The cuvettes are then transferred, one at a time, into the cell compart-</p><p>ment of a recording spectrophotometer, thermostated at 38". In rapid</p><p>succession DCIP and PMS (prewarmed to the assay temperature in red glass</p><p>test tubes) are added to initiate the reaction, and the absorbance decrease at</p><p>600 nm is recorded. The amount of enzyme used is so selected that the</p><p>absorbance decreases 30 to 50% of the chart width in 15 to 30 sec, thus</p><p>permitting measurement of the initial velocity. The absorbance range of the</p><p>recorder is normally 0.5 or 0.25 absorbance unit full scale; a t higher PMS</p><p>concentrations 10 to 12 in./min and at the lowest concentration 5 to 6</p><p>in./min chart speeds are recommended. Activity is then calculated from the</p><p>absorbance decrease, using the millimolar extinction coefficient for DCIP of</p><p>19.1 at 600 nm..</p><p>The measured rate is independent of the DCIP concentration under the</p><p>experimentalconditions until close to half of the dye is reduced, but it is</p><p>DETERMINATION OF THE ACTIVITY OF DEHYDROGENASES 135</p><p>greatly dependent on the concentration of PMS, which serves in this assay as</p><p>a catalyst between the dehydrogenase and DCIP (Figure 2). Hence, results</p><p>are usually expressed at V,, (PMS), except in certain types of routine work</p><p>(cf. below). Ideally, the PMS concentration should be varied over a 10-fold</p><p>range to yield a reliable slope in double reciprocal plots. With some sources</p><p>of the enzyme this is readily accomplished by varying the volume of PMS</p><p>used between 0.05 and 0.5 m1/3 ml reaction mixture. Some enzyme prep-</p><p>arations, however, are inhibited by 0.5 ml PMS/3 ml, while at concentra-</p><p>tions below 0.05 m1/3 ml the rate is usually too slow for accurate determina-</p><p>tion. Hence in practice the volumes of 0.33% PMS added per cell are as</p><p>follows: 0.30, 0.15, 0.10, 0.08, 0.06, and 0.05.</p><p>The blank rate (DCIP reduction in the absence of succinate) is negligible</p><p>with purified preparations but may be significant with particles, particularly</p><p>those containing very little succinate dehydrogenase. In such cases the blank</p><p>must be determined for each PMS concentration and subtracted.</p><p>SPECTROPHOTOMETRIC</p><p>PHENAZINE-DCPIP ASSAY</p><p>2 4 6 8 1 0</p><p>'/MG. PHENAZINE METHOSULFATE</p><p>Preactivated K:H. Preparation ; 50mM PO:,pH 7.6;</p><p>20 rnM Succinate; 9 m M CSA : I m u KCN ; I in 3 ml. Volume ; Temp. 3O'C.</p><p>Figure 2. Spectrophotometric determination of succinate dehydrogenase activity with PMS and</p><p>DCIP. Abscissa, reciprocal concentration of PMS, expressed as milligrams dye/3 ml reaction</p><p>mixture; ordinate, reciprocal of initial rate of absorbance change at 600 nm/min. Enzyme,</p><p>Keilin-Hartree preparation, 0.1 mg protein per cuvette. CSA, cysteinesulfinic acid; K-H,</p><p>Keilin-Hartree preparation. Data from Arrigoni and Singer. (16).</p><p>136 THOMAS P. SINGER</p><p>C. COMMENTS</p><p>In intact mitochondria pentration of the dyes is rate limiting (14,16). In</p><p>order to overcome this, 1 to 2 p g of purified phospholipase A (39) or 10 yg</p><p>crude Naja naja venom is added 1 min before the end of the activation</p><p>period. In some animal tissues a combination of 0.75mM CaCl, and cobra</p><p>venom may be required for free permeability; in such instances HEPES</p><p>buffer, pH 7.5, is substituted for phosphate to prevent precipitation of the</p><p>Ca2+.</p><p>When the activation properties of the enzyme are studied, the assay is</p><p>conducted at 15", where activation by succinate is extremely slow (25). In</p><p>such cases the activation phase is still carried out at 30 to 38", however, and</p><p>the cuvettes are then cooled for 3 min at 15" before adding the dyes.</p><p>Although the measurement of V,, is time consuming (about 20 min in</p><p>the hands of an experienced techincian), it is absolutely necessary in kinetic</p><p>studies, in comparisons of the activity or turnover number of the enzyme in</p><p>different tissues, in studying the effect of inhibitors, and even in calculating</p><p>yields on extraction and fractionation, since in all these procedures the</p><p>apparent K, for PMS may change. Hence results based on a single dye</p><p>concentration may yield misleading or even meaningless results (2,3,40). On</p><p>the other hand, when it is clear that a given procedure does not change the</p><p>K,,, for PMS, single-point assays (0.3 ml of PMS/3 ml) may be used in</p><p>routine work. Examples of this are chromatography of the enzyme on</p><p>Sephadex gels and activation-deactivation of the enzyme by various agents.</p><p>Occasionally assays carried out at 15" show a break in double reciprocal</p><p>plots at or below 0.08 ml of PMS/3 ml reaction mixture. Although the</p><p>reason for this has not been determined, it is likely to be the result of enzyme</p><p>modification. In such cases activity at V,, is calculated only from points</p><p>corresponding to the range 0.3 to 0.08 ml of PMS. Admittedly, this con-</p><p>centration range is somewhat narrow for accurate extrapolation.</p><p>Some users of the method routinely add 20 to 25 yl of unneutralized 0.1M</p><p>KCN just before the dyes in order to make up for cyanide which may have</p><p>escaped during activation at higher temperatures (e.g., 38"). Although this</p><p>precaution may not be necessary if the cuvettes are adequately covered</p><p>during activation and temperature equilibration, its inclusion does not</p><p>interfere with the assay.</p><p>King (41) reported that in his hands the spectrophotometric PMS-DCIP</p><p>method gave 15% variation in the extrapolated V,, value and the curves</p><p>relating enzyme concentration to measured activity did not extrapolate to</p><p>the origin. These criticisms are contrary to over 10 years' experience in this</p><p>laboratory and others (21) with the method. It may be noted in connection</p><p>with King's results that the intrument used is ill suited for kinetic work and</p><p>DETERMINATION OF THE ACTIVITY OF DEHYDROGENASES 137</p><p>that no steps were taken to activate the dehydrogenase. With some ex-</p><p>perience a single V,, determination should yield a value accurate to within</p><p>5%.</p><p>Alternative Procedures. Mammalian cytochrome c (5 X 10-5M) may be</p><p>substituted for DCIP in the assay in most cases (16). The manometric PMS</p><p>assay described in our earlier review (1) may be substituted, provided that</p><p>the assay temperature is below 30" or 100% 0, is used in the gas phase;</p><p>otherwise the rate of reoxidation of reduced PMS becomes rate limiting.</p><p>Substitution of a Clark 0, electrode for the manometric method entails the</p><p>same problem but to an even greater extent, because in this case the</p><p>dissolved 0, content falls to the point where the rate of reduction of the dye</p><p>exceeds its rate of reoxidation soon after initiation of the reaction. Hence use</p><p>of an 0, electrode for the assay of the succinate-PMS reaction is not</p><p>recommended.</p><p>5. Ferricyanide Assay</p><p>A. PRINCIPLE</p><p>The reduction of ferricyanide by succinate and succinate dehydrogenase is</p><p>monitored spectrophotometrically.</p><p>B. METHOD</p><p>REAGENTS. Potassium phosphate buffer, 0.3M, pH 7.5, at room tempera-</p><p>Sodium succinate, 0.2M, adjusted to pH 7.5 at room tem-</p><p>Potassium cyanide, 0.01 M, neutralized, prepared as in the</p><p>Potassium ferricyanide, 0.1M and O.OlM, kept in a dark</p><p>ture.</p><p>perature.</p><p>PMS-DCIP procedure.</p><p>bottle.</p><p>Procedure. A series of spectophotometer cuvettes are prepared to permit</p><p>varying the ferricyanide concentration. In each cuvette is placed 0.5 ml of</p><p>phosphate buffer, 0.3 ml of succinate, an amount of enzyme to give 0.2 to 0.5</p><p>optical density change per minutes at the highest ferricyanide cohcentration,</p><p>water to give a total volume of 3 ml during the assay, and 0.3 ml of cyanide.</p><p>The cuvettes are covered and activated at 30" or 38" as in the PMS-DCIP</p><p>assay. The cuvettes are transferred to the thermostated compartment of the</p><p>spectrophotometer, and the reaction is initiated by the addition of fer-</p><p>ricyanide.</p><p>138 THOMAS P. SINGER</p><p>$ 1.0</p><p>s</p><p>E</p><p>?!</p><p>\ 0.8</p><p>8 0.6</p><p>0</p><p>'u $ 0.4</p><p>P</p><p>$ 2 0.2</p><p>t</p><p>r) a</p><p>';.</p><p>50 100 150</p><p>l/ML 0.1 M FERRICYANIDE</p><p>5 10 15</p><p>I/ML 0.33% PMS</p><p>Figure 3. Comparison of PMS-DCIP and ferricyanide assays for succinate dehydrogenase. The</p><p>enzyme was extracted at pH 10.3 from a beef heart mitochondria1 acetone powder (37) and</p><p>precipitated with 0.46 sat. (NH,)$SO,. Assays were made after 6-min activation with succinate</p><p>at 38" at the temperatures specified, by the methods of Section 11.4 and 11.5. Unpublished data</p><p>from this laboratory.</p><p>The wavelength at which the reaction is followed depends on the amount</p><p>of ferricyanide used. In the range of 1.7 to 10 mM ferricyanide the</p><p>wavelength is 450 nrn and the millimolar extinction coefficient 0.262; below</p><p>this concentration 420 nm is used and the coefficient is 1.0. The range of</p><p>ferricyanide concentrations used is wider than in the PMS assay, because at</p><p>30 to 38" (and sometimes even at 25") a break occurs in double reciprocal</p><p>plots relating optical density change to ferricyanide concentration (Figure</p><p>3). The amounts of ferricyanide solution to be added for the determination</p><p>of VmM are as follows: 0.3, 0.2, 0.15, 0.1, 0.08, and 0.05 ml of 0.1M</p><p>ferricyanide. The biphasic curve (cf. below) is seen at lower concentrations</p><p>of ferricyanide (0.2, 0.15, 0 . 1 , and 0.08 ml of 0.01M ferricyanide). Absor-</p><p>bance change is measured with the same instrument as is recommended for</p><p>the CoQ reductase assay; the scale expansion (range) and chart speed are</p><p>adjusted so as to give a tracing between the 30 and 60" angles. The blank</p><p>(no succinate) is negligible with purified preparations. In crude preparations</p><p>the amount of bleaching in the absence of succinate must be determined</p><p>and, if significant, subtracted from the experimental values. In calculating</p><p>DETERMINATION OF THE ACTIVITY OF DEHYDROGENASES 139</p><p>1 I I I</p><p>1/ML FERRICYANIDE</p><p>10 20 30 40 5c</p><p>I I I I I I I I</p><p>2 4 6 8 10 12 14 16</p><p>1/ML PMS</p><p>Figure 4. Comparison of PMS-DCIP and ferricyanide assays at 25". The enzyme was an</p><p>extract of acetone powder or beef heart ETP. Before assay, the enzyme was activated for 5 min</p><p>at 38" with succinate. Crosses, ferricyanide assay; shaded circles, PMS assay. Unpublished data</p><p>from this laboratory.</p><p>the results the fact that 1 mole of succinate reduces 2 moles of ferricyanide is</p><p>taken into consideration.</p><p>With intact mitochondria purified phospholipase A or crude N. naja</p><p>venom should be added in the amounts specified for the PMS-DCIP assay,</p><p>in order to permit free penetration of the ferricyanide.</p><p>C. COMMENTS</p><p>As may be seen in Figure 3, at temperatures above 25" double reciprocal</p><p>plots are biphasic in the ferricyanide assay. In fact, the data illustrated are</p><p>suggestive of a possible biphasic plot even at 25". The break in the curve is</p><p>not due to a second reaction site at the cytochrome c- c1 level, since</p><p>cytochromes are not present in the purified enzyme; moreover, the same</p><p>break may be seen in particles inhibited with antimycin A. The main reason</p><p>why this biphasic curve has been overlooked by others is that assays in other</p><p>laboratories are based either on a single ferricyanide concentration or on a</p><p>series of concentrations in a limited, high range, so that the break in the</p><p>curve escapes detection. From the steepness of the curve it is also evident</p><p>140 THOMAS F. SINGER</p><p>that assays at a fixed ferricyanide concentration (41,42) may yield mislead-</p><p>ing results.</p><p>The data in Figure 3 also show that at 38" the ferricyanide assay at Vm,,</p><p>measures a much smaller fraction of the activity than does the PMS assay.</p><p>At 25" the deviation is less and sometimes (Figure 4) absent (21).</p><p>As seen in Figure 3, concentrations of ferricyanide as high as 10 mM do</p><p>not seem to inhibit the enzyme. This is contrary to the experience of Veeger</p><p>et al. (42) that concentrations in excess of 5 mM are inhibitory.</p><p>An obvious source of major error in the method is the use of different</p><p>wavelengths at different ferricyanide concentrations. At 450 nm the absorp-</p><p>tion curve of ferricyanide declines extremely steeply with increasing wave-</p><p>length, so that a 1-nm deviation in the wavelength setting or the use of a</p><p>wide slit width can cause significant error. The problem may be circum-</p><p>vented in two ways. One may conduct all measurements at the longer</p><p>wavelength if the spectrophotometer used performs reliably at a scale</p><p>expansion of 0.1 or 0.05 absorbance unit full scale. Alternatively, the</p><p>absorbance of a standard ferricyanide solution may be read at each wave-</p><p>length used immiediately after the assay and the extinction coefficient of</p><p>ferricyanide at the longer wavelength empirically determined from the fixed</p><p>value of E~ = 1 .O at 420 nm.</p><p>6. Fumarate Reductase Assay</p><p>A. PRINCIPLE</p><p>The assay is based on the reduction of fumarate by FMNH, and the</p><p>regeneration of the latter by H, and hydrogenase. The H, uptake is</p><p>measured manometrically.</p><p>B. METHOD</p><p>REAGENTS. Phosphate buffer, 0 .3M, pH 7.6.</p><p>Fumarate, l M , pH 7.6.</p><p>FMN,5x 10-3M (aqueous solution, protected from light in a</p><p>dark bottle and kept frozen when not in use).</p><p>Wydrogenase. Clostridium pasteunanum, strain W5, is carried as</p><p>a spore suspension. Subcultures are first grown at 37" in Brewer</p><p>jars under illuminating gas in the following medium: 2% glu-</p><p>cose, 0.1 % tryptone, 0.1 % agar, and 0.2% CaCO,. When growth</p><p>is evident from gas formation (24 to 48 hr), transfer is made to</p><p>test tubes containing 1 % glucose (sterilized separately), 0.4%</p><p>yeast extract, 0.1% beef extract, 1.25% K,HPO,, and 10% (v/v)</p><p>tap water. The medium is adjusted to pH 7.0 with 5N HCl.</p><p>DETERMINATION OF THE ACTIVITY OF DEHYDROGENASES 141</p><p>Vigorous growth should be evident after 12 hr at 37" (in</p><p>Brewer jars under illuminating gas), and at that time a 5%</p><p>(v/v) inoculum is made into Erlenmeyer flasks, each contain-</p><p>ing 100 ml of the same medium. After about 12 hr of growth at</p><p>37" under N, or illuminating gas, the content of one flask is</p><p>used as inoculum for 8 to 10 liters of the same growth medium.</p><p>The organisms may be conveniently grown in 12-liter Florence</p><p>flasks (10 liters of medium) or 5-gal bottles (16 liters of</p><p>medium). After inoculation the medium is gassed aseptically</p><p>through a sparger with N, until vigorous H, evolution is</p><p>evident and is then maintained at 37". If the anaerobiosis is</p><p>satisfactory, heavy growth occurs in 12 to 18 hr. After cooling</p><p>the suspension below room temperature, the organisms are</p><p>collected in a refrigerated Sharples centrifuge. Lots of 25 g are</p><p>ground in an ice-cold mortar with 62.5 g of alumina (Alcoa,</p><p>A-301). When the suspension turns to a thick, muddy paste (2</p><p>to 3 min), 50 ml of glass-distilled H,O is stirred in gradually,</p><p>and the suspension is immediately centrifuged at 10,000 g for</p><p>15 to 20 min so as to yield brown, translucent, but not complete&</p><p>clear, supernatant solution, which is frozen in test tubes under</p><p>H, and may be preserved for years at - 20".</p><p>The bacterial extract thus obtained possesses a powerful</p><p>hydrogenase; it is devoid of succinate dehydrogenase and</p><p>should give no H, uptake if either fumarate of succinate</p><p>dehydrogenase is omitted. The activity of each batch is checked</p><p>by varying the amount of hydrogenase at fixed succinate de-</p><p>hydrogenase and FMN concentrations (0.4 ml of 5 X 10-3M</p><p>FMN/3 ml). Usually 0.2 to 0.3 ml of extract provides a</p><p>sufficient excess for the assay described below. Useful criteria of</p><p>a satisfactory preparation are that it should bleach the fluores-</p><p>cence of FMN within a few seconds after the addition of</p><p>hydrogenase to the vessel, and that if 1 X 10-3M methyl violo-</p><p>gen is substituted for FMN, almost immediate formation of a</p><p>deep purple color should occur on addition of hydrogenase.</p><p>Procedure. Each of a set of Warburg flasks (15- to 30-ml capacity)</p><p>contains 0.5 ml of phosphate buffer, enzyme, H,O to give a final volume of 3</p><p>ml, and varying amounts of FMN. For each enzyme sample five to six</p><p>vessels are used,</p><p>and the amount of dye is varied between 0.4 and 0.1 ml per</p><p>vessel. The side arm, equipped with a vented plug, receives 0.1 ml of</p><p>fumarate. The vessels are placed on their manometers and gassed with H,</p><p>for 3 min with periodic shaking. At that time hydrogenase (0.2 to 0.3 ml; see</p><p>142 THOMAS P. SINGER</p><p>above) is added rapidly to the main compartment, and the vessels are gassed</p><p>for an additional 5 min. The rapid addition of hydrogenase is facilitated if</p><p>the flask is equipped with a side opening like that in the Corning No. 5333</p><p>flask. Alternatively, a two-side-arm vessel may be used and the hydrogenase</p><p>tipped in from the second side arm during gassing.</p><p>After 15 min of equilibration at 38”, fumarate is added from the side arm,</p><p>and H, uptake is followed for 25 to 30 min. The rate should be linear with</p><p>time, except for a slight lag occasionally observed during the initial 5-min</p><p>period. From the linear rate the Vm, is calculated by the double reciprocal</p><p>method (1 /H, versus 1 /FMN).</p><p>In preparations possessing an intact mitochondria1 membrane the inclu-</p><p>sion of 0.75mM CaC1, is recommended in order to allow free penetration of</p><p>the dye, and HEPES buffer is substituted for phosphate to avoid the</p><p>precipitation of calcium phosphate.</p><p>7. “Reconstitution” Activity</p><p>A. PRINCIPLE</p><p>Exposure of respiratory chain preparations to pH 9.3 to 9.4 under suitable</p><p>conditions results in inactivation of succinate dehydrogenase with little</p><p>damage to other components. Soluble succinate dehydrogenase recombines</p><p>with such alkali-treated preparations with resulting recovery of succinoxi-</p><p>dase activity (43). The amount of succinate dehydrogenase taken up equals</p><p>the succinate dehydrogenase content before alkali treatment. Since the alkali</p><p>0.2 0.4 0.6 0.8 1.0</p><p>ML. SOLUBLE ENZYME</p><p>Figure 5. Regeneration of succinoxidase activity resulting from the recombination of a soluble</p><p>preparation of succinate dehydrogenase with an alkali-treated heart muscle preparation. From</p><p>Singer (2); experimental conditions as in Figure 6.</p><p>DETERMINATION OF THE ACTIVITY OF DEHYDROGENASES 143</p><p>treatment removes little, if any, succinate dehydrogenase, the reconstituted</p><p>complex shows approximately twice the original content of the enzyme, as</p><p>may be readily ascertained by analysis for histidyl-flavin (22,44). Only the</p><p>newly acquired flavoprotein is catalytically competent in the reconstituted</p><p>complex (2).</p><p>Not all soluble, purified preparations of the dehydrogenase are capable of</p><p>reconstituting succinoxidase activity; preparations isolated in the absence of</p><p>succinate may show very high catalytic activity but are inactive in the</p><p>reconstitution test (41). Some preparations which are inactive in this test are</p><p>reported to be reactivated on incubation with inorganic iron, sulfide, and</p><p>mercaptoethanol (45). Even “reconstitutively active” preparations may be</p><p>heterogeneous, containing at least three forms of the dehydrogenase: one</p><p>which combines with alkali-treated particles and confers succinoxidase activ-</p><p>ity, another which recombines without conferring succinoxidase activity, and</p><p>1.4</p><p>I. 2</p><p>1.0</p><p>0.8</p><p>0.6</p><p>0.4</p><p>0.2</p><p>0.05 0.10</p><p>BOUND FLAVIN INCORPORATED</p><p>mpMOLES / MG. PROTEIN</p><p>Figure 6. Relation between the incorporation of bound flavin (histidyl-flavin) and the reactiva-</p><p>tion of succinate oxidase (SO) and succinate dehydrogenase (SD). Aliquots of an alkali-</p><p>inactivated, neutralized Keilin-Hartree preparation (0.6 ml containing 18.1 mg protein) were</p><p>mixed at 0” with varying amounts (0.3 to 6.0 ml) of soluble succinic dehydrogenase in 0.075M</p><p>phosphate buffer, pH 7.8, prepared by the method of King and containing 3.77 mg protein/ml.</p><p>The mixtures were diluted to 6.6 ml with 0.075M phosphate, pH 7.8, centrifuged for 15 min at</p><p>144,000gmU; the supernatant solution was discarded, and the pellet washed twice with 6.6 ml of</p><p>the Same buffer by centrifugation. Aliquots of the untreated Keilin-Hartree preparation and of</p><p>the alkali-inactivated one, to which no dehydrogenase had been added, were similarly centri-</p><p>fuged and washed. Aliquots of the final pellet in each case were assayed for bound flavin,</p><p>succinate dehydrogenase, and succinoxidase. The values for the original sample were 0.129</p><p>mpmoles bound flavin/mg protein and 1.47 and 0.897 pmoles succinate oxidized/min/mg</p><p>protein in the dehydrogenase and oxidase assays, respectively. The corresponding figures for the</p><p>alkali-treated samples were 0.1 15 mpmoles/mg and 0.198 and 0.041 pmoles/min/mg. From</p><p>Kimura et al. (44).</p><p>144 THOMAS P. SINGER</p><p>a third which does not recombine with alkali-treated particles (22,46).</p><p>The procedure given below utilizes Keilin-Hartree particles as the source</p><p>of the cytochrome system but works equally well for ETP, Complex 11, and</p><p>other preparations derived from the inner mitochondria1 membrane. The</p><p>method entails the titration of an alkali-treated particle preparation with</p><p>soluble succinate dehydrogenase and measurement of the resulting restora-</p><p>tion of succinoxidase activity, as in Figure 5 (since Complex I1 has no</p><p>oxidase activity, in this case succinate-CoQ reductase activity may be used).</p><p>In order to ascertain what fraction of the added, soluble succinate de-</p><p>hydrogenase is “reconstitutionally active,” it is necessary to perform addi-</p><p>tional analyses and calculations. It is seen in Figure 6 that the amount of</p><p>histidyl-flavin incorporated in the particle is linearly related to the</p><p>succinoxidase and succinate dehydrogenase activities conferred upon the</p><p>particles by the incorporated dehydrogenase. Hence, if the reconstituted</p><p>particle is washed by centrifugation to remove uncombined, soluble</p><p>succinate dehydrogenase at any point in the titration curve (Figure 5) and</p><p>the succinoxidase (Section 11.2) or succinate-PMS (Section 11.4) activity of</p><p>the pellet is determined and divided by the bound flavin which was</p><p>incorporated, the turnover number per mole of newly acquired flavin may</p><p>be calculated. With alkali-treated ETP as a source of the cytochrome system</p><p>this figure should be the same as in untreated ETP (18,OOOk 1000 at 38”) in</p><p>the PMS assay. A lower value indicates incorporation of succinate de-</p><p>hydrogenase capable of recombination but not of conferring succinate de-</p><p>hydrogenase activity.</p><p>0 2 0</p><p>of the main</p><p>batch but carry out the pH adjustment a t Oo.> The sample is then either</p><p>incubated at 37" for 60 min or at 20 to 25" for 100 to 120 min, monitoring</p><p>in either case for decay of succinoxidase activity (cf. Section 11.2). When</p><p>95% loss of succinoxidase activity is reached, the preparation is rapidly</p><p>chilled to 0" and slowly neutralized to pH 7.4 to 7.6 with 1N acetic acid.</p><p>For reconstitution of the succinoxidase activity, buffer, cytochrome c, and</p><p>varying amounts of soluble, actzuated succinate dehydrogenase are placed in</p><p>the 0, electrode chamber (see the method of Section 11.2), and the reaction</p><p>is initiated by the addition of the alkali-treated Keilin-Hartree preparation.</p><p>The quantity of Keilin-Hartree preparation used is such as to give a</p><p>convenient rate of 0, uptake (e.g., 30 to 80% consumption of the dissolved</p><p>0, in 5 min) after full reactivation (or before alkali inactivation). The</p><p>amount of soluble enzyme added is varied between 0 and 1 nmoles of</p><p>histidyl-flavin added/mg of protein in the Keilin-Hartree preparation.</p><p>[Although an untreated Keilin-Hartree preparation contains only 0, l l to</p><p>0.14 nmoles histidyl-flavin/mg, with most "reconstitutively active" soluble</p><p>preparations several times this amount must be added to attain full restora-</p><p>tion of succinoxidase activity (47).] Since recombination of the soluble</p><p>enzyme with the particle is rapid even below room temperature, a linear</p><p>146 THOMAS P. SINGER</p><p>rate of 0, uptake will be observed after a few seconds’ lag, provided that the</p><p>soluble enzyme has been fully activated (cf. Figure 13 in ref. 2).</p><p>The resulting rate of 0, uptake may be plotted against the quantity of</p><p>soluble enzyme added, as in Figure 5, and compared with the rate given by</p><p>a sample of the Keilin-Hartree preparation before alkali treatment, so as to</p><p>ascertain the fraction of the activity inactivated by alkali which has been</p><p>restored. In order to determine the fraction of soluble succinate de-</p><p>hydrogenase capable of recombination with the alkali-treated heart muscle</p><p>preparations, recombination is performed in the cold, uncombined succinate</p><p>dehydrogenase is removed by centrifugation, and the histidyl-flavin content</p><p>is determined at each level of added succinate dehydrogenase, as specified in</p><p>the legend of Figure 7.</p><p>8. Determination of Succinate Dehydrogenase by Analysis</p><p>for Covalently Bound Flavin</p><p>A. PRINCIPLE</p><p>The fluorometric determination of covalently bound flavin, first developed</p><p>by Kearney (48,49) has been described in detail (4,35); therefore the</p><p>present review discusses only its principles and limitations.</p><p>The concentration of succinate dehydrogenase in tissues and enzyme</p><p>preparations may be determined by analysis of the histidyl-flavin content</p><p>(4,34). The method is applicable only to samples in which succinate</p><p>dehydrogenase is definitely known to be the only source of histidyl-flavin.</p><p>This has been established for heart and aerobic yeast mitochondria, as well</p><p>as preparations derived from these sources, and appears to be also true for</p><p>brain mitochondria. In liver mitochondria, however, a considerable fraction</p><p>of the covalently bound (i.e., acid-nonextractable) flavin is associated with</p><p>sarcosine dehydrogenase (50), and the pH-fluorescence curves of peptides</p><p>isolated from this enzyme suggest that it may also contain histidyl-flavin</p><p>(5 1). Since sarcosine dehydrogenase is a matrix enzyme, whereas succinate</p><p>dehydrogenase is located in the inner membrane, in purified preparations of</p><p>the inner membrane of liver mitochondria, or extracts thereof, histidyl-flavin</p><p>may be a valid measure of succinate dehydrogenase content.</p><p>Before the identification of histidyl-8a-FAD in succinate dehydrogenase</p><p>(52), this compound was referred to simply as “covalently bound flavin” in</p><p>the literature. By now two other types of covalently bound flavin have been</p><p>identified and isolated, in addition to histidyl-flavin (53). Although these are</p><p>also substituted at the 8a-position of riboflavin, the substituent in these cases</p><p>is cysteine, not histidine, and thus the characteristic variation of the fluores-</p><p>cence with pH (a consequence of the protonation of the imidazole nitrogen)</p><p>is absent in these compounds. Thus proteolytic digests of enzymes containing</p><p>DETERMINATION OF THE ACTIVITY OF DEHYDROGENASES 147</p><p>cysteinyl-flavin thioether of thiohemiacetal (53) show the same fluorescence</p><p>at pH 3.2 and 7.0, while histidyl-flavin exhibits only 5 to 10% as much</p><p>fluorescence at pH 7.0 as at pH 3.2 to 3.4. Subtraction of the fluorescence at</p><p>pH 7.0 from that measured at pH 3.2 to 3.4 therefore permits the determina-</p><p>tion of histidyl-flavin in the presence of contaminating cysteinyl-flavin. The</p><p>membrane fractions of brain and liver mitochondria, for instance, contain</p><p>significant amounts of cysteinyl-riboflavin originating from monoamine</p><p>oxidase (54,55), but the fluorescence difference at the two pH values</p><p>nevertheless permits quantitative estimation of their histidyl-flavin contents.</p><p>A more serious problem is the presence of histidyl flavin of the succinate</p><p>dehydrogenase type originating from other enzymes, as, for instance, the</p><p>flavin of sarcosine dehydrogenase in liver mitochondria. In such cases</p><p>histidyl flavin content cannot be equated with succinate dehydrogenase, of</p><p>course. Recently another type of histidyl flavin has been discovered in</p><p>thiamine dehydrogenase (55a) and P-cyclopiazonate oxidocyclase (55b),</p><p>from microorganisms, in which the imidazole appears to be linked to</p><p>8-formyl FAD. This type of flavin would be codetermined in the method</p><p>given below, but it has not yet been found present in animal tissues.</p><p>B. METHOD</p><p>The method (4) involves four steps. First, the acid-extractable flavin is</p><p>removed by repeated extractions with trichloroacetic acid. Second, the</p><p>residue is digested with suitable proteolytic enzymes (usually trypsin plus</p><p>chymotrypsin) in order to render the covalently bound flavins water soluble.</p><p>Insoluble peptides are then removed by precipitation with trichloroacetic</p><p>acid. Third, the resulting extract is acid hydrolyzed in order to convert</p><p>flavin dinucleotides to the FMN level. (In FAD and its derivatives the</p><p>fluorescence is some 85% quenched by the adenylate moiety at pH 7, which</p><p>would interfere with measurement of the variation of fluorescence with pH.)</p><p>Fourth, the fluorescence of the sample is measured at both pH 3.2 to 3.4 and</p><p>pH 7.0 before and after the addition of hydrosulfite, with and without added</p><p>riboflavin as internal standard. Residual fluorescence after hydrosulfite is</p><p>used as blank to correct for nonflavin fluorescence, while the internal</p><p>standard serves to correct for quenching of flavin fluorescence by impurities</p><p>in the sample. The difference in flavin fluorescence at the two pH values,</p><p>corrected for this quenching, is used to calculate the histidyl-flavin content.</p><p>C. COMMENTS</p><p>Although the method is relatively simple and is reproducible with purified</p><p>enzyme preparations, serious difficulties may arise in its application to crude</p><p>material, particularly if the succinate dehydrogenase content is very low.</p><p>Thus in membrane preparations five to ten extractions with trichloracetic</p><p>148 THOMAS P. SINGER</p><p>acid may be required to remove all acid-extractable flavin. In certain plant</p><p>mitochondria the nonflavin fluorescence (i.e., fluorescence not quenched by</p><p>hydrosulfite) may be very high, with resultant excessive :'blanks." In samples</p><p>containing relatively large amounts of covalently bound flavin other than</p><p>histidyl-flavin the fluorescence at pH 7.0 approaches that measured at pH</p><p>3.2 to 3.4. As a result a relatively small error in fluorometry may cause a</p><p>large error in calculating the difference in fluorescence at the two pH values.</p><p>This is the case in submitochondrial preparations enriched in the outer</p><p>membrane fraction (because of monoamine oxidase), in anaerobic yeast, and</p><p>in yeast mitochondria during the early stages of mitochondrial biogenesis.</p><p>111. APPLICATION OF SUCCINATE</p><p>DEHYDROGENASE ASSAYS TO YEAST, BACTERIA,</p><p>AND HIGHER PLANTS</p><p>The methods of succinoxidase and succinate dehydrogenase assay used for</p><p>Saccharomyces cerevisiae preparations in the author's laboratory (56) are minor</p><p>variations of the procedures detailed in Section II for animal tissues, since</p><p>the properties of the enzyme in aerobic yeast and animal tissue are very</p><p>similar. In the succinoxidase assay 50mM succinate is used during assay,</p><p>which is routinely carried out at 30".</p><p>In the PMS-DCIP assay the buffer is 40mM phosphate, pH 7.5; both</p><p>activation and assay are carried out at 30" and activation comes to cornple-</p><p>tion in 7 min at 30" in the presence of -25rnM succinate and the amount of</p><p>mitochondria1 or soluble enzyme which gives rates comparable to those</p><p>specified in Section 11. An important precaution is to run blanks (no</p><p>succinate) with yeast samples which are not fully aerobic, since in these</p><p>instances reduction of the dyes by endogenous substrates may be appreciable</p><p>T h e conditions and reagents for measurement of the succinate-</p><p>ferricyanide activity of yeast succinate dehydrogenase are the same as those</p><p>for the PMS-DCIP assay, except that ferricyanide is used as terminal</p><p>oxidant. Since in yeast mitochondria and subrnitochondrial particles from</p><p>aerobic yeast the Lineweaver-Burk plot in the ferricyanide assay is biphasic</p><p>(56), it is necessary to measure the activity at a large number of ferricyanide</p><p>concentrations in calculating Vmax [Fe(CN,)-3]. In a 3-ml final volume the</p><p>following amounts of 0.01M ferricyanide are used: 0.50, 0.3, 0.25, 0.2, 0.16,</p><p>0.125, 0.10, 0.08, and 0.07 ml.</p><p>It has been reported (57) that the reaction of yeast succinate de-</p><p>hydrogenase is slower with PhlS than with 0, via the respiratory chain.</p><p>This finding appears to be erroneous (cf. Table I) and may have been due to</p><p>the use of fixed PMS concentration (rather than measurement of V,,,), an</p><p>(56).</p><p>DETERMINATION OF THE ACTIVITY OF DEHYDROGENASES 149</p><p>TABLE I</p><p>Comparison of Succinate Dehydrogenase Assays</p><p>in Submitochondrial Particles from Aerobic Yeast</p><p>Amount of succinate</p><p>oxidized ( pmoles/min. mg</p><p>k Y of protein)</p><p>Preparation No. 1 2</p><p>S u cc i n o xi d ase 1.24 1.13</p><p>PMS-DCIP 1.76 1.80</p><p>DCIp6 0.11 0.12</p><p>Femcyanide" 0.30</p><p>~~ ~</p><p>"V,, with respect to dye.</p><p>'Fixed dye concentration (conditions as per ref.</p><p>58). All assays were at 30".</p><p>assay temperature a t which even membrane-bound succinate dehydrogenase</p><p>from yeast is unstable, or failure to activate the enzyme.</p><p>Succhuromyces cerevisiue also contains cytoplasmic fumarate reductases which</p><p>reduce fumarate to succinate quite rapidly but do not oxidize succinate to</p><p>fumarate at measurable rate with any known electron acceptor (59). There</p><p>are several isoenzymes of yeast fumarate reductase, which fall into two</p><p>classes. The two types differ not only in molecular weight and charge but</p><p>also in catalytic properties, such as their reactivities with electron donors</p><p>(Figure 8). With both types of enzyme the highest activity is measured with</p><p>B.</p><p>M.V.</p><p>FMNH,</p><p>B .V.</p><p>2 4 6 2 4 6 8</p><p>I</p><p>DYE C O X . (mM)</p><p>Figure 8. Comparison of specificities of fumarate reductase isoenzymes type I (A) and type 11</p><p>(E) for electron donors. M. V., methylviologen; E . V., benzylviologen. All assays were at 30°,</p><p>pH 7.6, by manometric measurement of hydrogen uptake in the presence of excess hydrogenase</p><p>from Cl. partcurianurn. Modified from Hauber and Singer (59).</p><p>150 THOMAS P. SINGER</p><p>FMNH, as reductant, and the rate does not vary with dye concentration</p><p>(Figure 8). Hence these enzymes may be assayed by the procedure given in</p><p>Section 11.6, but using a fixed (1mM) FMN concentration.</p><p>With few exceptions, methods for the assay of bacterial succinate de-</p><p>hydrogenases have been inadequately explored. Depending on the species</p><p>and growth conditions, bacterial cells may contain a typical aerobic-type</p><p>succinate dehydrogenase, an anaerobic one, such as the cytoplasmic enzyme</p><p>in yeast (60), or both (61). In the case of the anaerobic enzyme from</p><p>Micrococcus lactilyticus, perhaps the best-characterized one in this group</p><p>(60,62), succinate oxidation may be measured with the standard PMS-</p><p>DCIP assay but in 0.05M Tris buffer, pH 8.5, at 30" without activation,</p><p>while the much higher fumarate reductase activity is assayed by the method</p><p>of Section 11.6 at 30", using 0.01M fumarate, 0.05M phosphate buffer, pH</p><p>7.5, and varying amounts of methyl viologen (0.3 to 2mM), with calcula-</p><p>tions based on V,, with respect to the dye (60). Succinate dehydrogenase</p><p>from Propiombacterium pentosaceurn (63) in both membrane-bound and soluble</p><p>form may be assayed by the standard PMS-DCIP method (Section 11.4) but</p><p>at pH 7.4 and 30". Activation is not known to be required.</p><p>The following variation of the procedure of Section 11.2 has been used for</p><p>assay of the succinoxidase activity of mitochondria from higher plants</p><p>(cauliflower, mung bean) in this laboratory (64). Activation is performed</p><p>either in open test tubes with malonate or anaerobically in Thunberg tubes</p><p>with succinate as activator (method of Section 11.2). An aliquot (0.04 to 0.05</p><p>ml, containing 0.8 to 1.2 mg of mitochondria1 protein) is then added to the</p><p>0, electrode chamber ( 1 . 8 4 cell), thermostated at 30" and containing 1.74</p><p>ml of a solution consisting of 0.3M mannitol- lOmM KCl-5mM MgC1,-</p><p>lOmM Nap,, adjusted to pH 7.2. With cauliflower mitochondria bovine</p><p>serum albumin is also present at 1 mg/ml concentration. The inclusion of</p><p>cytochrome c does not seem necessary with good-quality mitochondria. The</p><p>reaction is then started by the addition of 0.01 ml of 1.6M succinate. State</p><p>4+3 transition is initiated with 5 p1 0.04M ADP.</p><p>The succinate dehydrogenase activity of cauliflower or mung bean mi-</p><p>tochondria and submitochondrial particles is measured by an adaptation of</p><p>the method of Section 11.4 (64). In a 3-ml final volume 0.5 ml of 0.2M</p><p>HEPES buffer, pH 7.5, is substituted for phosphate. Assays are carried out</p><p>at 25 to 30", except in studies involving activation of the enzyme, where the</p><p>assay temperature is 15". For routine assays a fixed PMS concentration (0.3</p><p>ml of 0.33%, W/V) is used; for accurate work V,, with respect to PMS is</p><p>determined.</p><p>Since the enzyme in cauliflower mitochondria is very unstable in the</p><p>absence of thiols but may be preserved for prolonged periods in media</p><p>containing 1mM cysteine, precautions are taken to both minimize and</p><p>correct for the blank reduction of PMS and DCIP by the added thiol. This</p><p>DETERMINATION OF THE ACTIVITY OF DEHYDROGENASES 151</p><p>is achieved by adding 0.01 ml of 0.03M N-ethylamaleimide (NEM) just</p><p>before the 3-min temperature equilibration preceding the assay. This allows</p><p>sufficient time for combination of the cysteine with NEM. In addition, with</p><p>each series of experiments a blank is run to correct for spurious dye</p><p>reduction by performing the assay under identical conditions except for the</p><p>ommission of succinate. The resulting rate of dye reduction is then sub-</p><p>tracted from each experimental value.</p><p>Activation is performed with succinate by incubation of all assay con-</p><p>stituents, less NEM, PMS, and DCIP, for 8 min at 30", as in the case of the</p><p>mammalian enzyme. Then NEM is added, the cuvettes are equilibrated for</p><p>3 min at the temperature of the assay, and the reaction is started by rapid</p><p>addition of PMS and DCIP. Alternatively, the enzyme may be activated in</p><p>batches by incubation for 12 to 15 min at 30" with 0.1M succinate in the</p><p>presence of 1mM cyanide at 2 to 6 mg protein concentration/ml. A small</p><p>aliquot is then placed directly into the assay cuvette, containing all assay</p><p>constituents, including NEM, but not PMS or DCIP, and temperature</p><p>equilibrated for 3 min; the assay is started by the addition of the dyes.</p><p>Activation is absolutely essential with the cauliflower enzyme, which is</p><p>normally obtained 80 to 95% in the deactivated state in mitochondria. In</p><p>mung bean mitochondria</p>