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Some Properties of a Detergent-solubilized 
NADPH-Cytochrome c (Cytochrome P-450) Reductase Purified 
by Biospecific Affinity Chromatography* 
(Received for publication, March 22, 1976) 
YUKIO YASUKOCHI AND BETTIE SUE SILER MASTERS 
From the Department of Biochemistry, The University of Texas Health Science Center at Dallas, 
Southwestern Medical School, Dallas, Texas 75235 
NADPH-cytochrome c (cytochrome P-450) reductase (EC 1.6.2.4) has been purified to homogeneity, as 
judged by sodium dodecyl sulfate disc gel electrophoresis, from detergent-solubilized rat and pig liver 
microsomes using an affinity chromatography procedure. Treatment of microsomes with a polyethox- 
ynonylphenyl ether plus either cholate or deoxycholate and subsequent batch-wise DEAE-cellulose 
chromatography followed by biospecific affinity chromatography on Sepharose 4B-bound N6- 
(6-aminohexyl)adenosine 2’,5’-bisphosphate (2’,5’-ADP-Sepharose 4B) result in a > 30% yield of purified 
reductase from microsomes. The enzyme contains 1 mol each of FAD and FMN and exhibits a molecular 
weight of 78,000 g mall’ estimated by comparison with protein standards on sodium dodecyl sulfate 
polyacrylamide gel electrophoresis. The turnover numbers calculated on the basis of flavin are 1360 
min’ and 1490 min-’ at 25” for the pig and rat liver enzymes, respectively. 
Titration of these purified preparations aerobically with both NADPH and potassium ferricyanide 
demonstrated unequivocally that the air-stable, reduced form of NADPH-cytochrome c (P-450) 
reductase contains 2 electron equivalents, confirming recent results obtained by Masters et al. (Masters, 
B. S. S., Prough, R. A., and Kamin, H. (1975) Biochemistry 14, 607-613) for the proteolytically 
solubilized enzyme. In addition, these preparations are capable of reconstituting benzphetamine 
N-demethylation activity in the presence of partially purified cytochrome P-450 and dilauroylphos- 
phatidylcholine, as measured by formaldehyde formation from benzphetamine. 
Numerous methods for the purification of hepatic micro- 
somal constituents have been employed resulting in homogene- 
ous preparations of NADPH-cytochrome c (P-450) reductase 
(1, 2) and cytochrome P-450 (3-5). Various detergents have 
been utilized, but the most successful combinations have 
included the polyethoxyalkylaryl ethers, such as Emulgen 911 
(3) or Renex 690 (1, 2, 4, 5). The use of such detergents has 
produced preparations which are capable of reconstituting 
microsomal hydroxylation reactions. A recent report by Golf et 
al. (6) described the use of an affinity chromatography column 
consisting of cytochrome c bound to Sepharose 4B, but the 
final preparation was not homogeneous and the enzyme could 
be eluted upon the addition of 1 M KC1 to the equilibration 
buffer. It has been, however, only with great difficulty that 
purification to homogeneity has been achieved with any of 
these preparations and the multiple steps required have 
resulted in low percentage yields (1, 2). 
The present paper will report purification procedures for rat 
and pig liver NADPH-cytochrome c (P-450) reductase which, 
in three and four chromatographic steps, respectively, from 
solubilized microsomes produce spectrally pure enzyme prepa- 
* This work was supported by United States Public Health Service 
Grants HL13619 and GM16488 and by Grant I-453 from The Robert A. 
Welch Foundation. 
rations capable of reconstituting benzphetamine metabolism 
with a partially purified preparation of cytochrome P-450 and 
dilauroylphosphatidylcholine. The procedure involves the use 
of Sepharose 4B-bound Ne-(6..aminohexyb-adenosine 2’,5’-bis- 
phosphate prepared by the method of Brodelius et al. (7). 
Recent reports from this laboratory (8-10) confirmed the 
earlier results obtained with proteolytically solubilized reduc- 
tase demonstrating 2 electron equivalents in the air-stable, 
half-reduced form of the reductase. The present results indi- 
cate that the detergent-solubilized NADPH-cytochrome c 
(P-450) reductase exhibits identical spectral properties with 
the proteolytically solubilized flavoprotein. Aerobic titration 
with NADPH to the air-stable, reduced form of the enzyme 
and back titration with potassium ferricyanide to the fully 
oxidized state reveals that it, too, contains 2 electron equiva- 
lents, in agreement with previous results from this laboratory 
(8-12) but in contrast to the reports of Iyanagi et al. (13-15). 
EXPERIMENTAL PROCEDURE 
Preparation of Pig Liver Microsomes-A pig liver homogenate was 
prepared according to the procedure of Masters et al. (16) in a ratio of 1 
g of liver/3 ml of 0.25 M sucrose, pH 7.7 (25% w/v). The supernatant 
from centrifugation of this homogenate at 11,320 x g (R,.,) in the 
Beckman J-21B was diluted 2.5.fold with cold distilled water and 
mixed with a CaCl, solution yielding a final concentration of 8 mM, 
5337 
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5338 Affinity-chromatographed NADPH-Cytochrome c (P-450) Reductase 
according to the method of Cinti et al. (17). This mixture was 
centrifuged at 17,680 x g for 60 min. The microsomal pellet was 
washed by suspending in 0.1 M sodium pyrophosphate buffer, pH 7.4, 
containing 1 rnM EDTA, and centrifuging in the Spinco L2-65B at 
105,000 x g for 60 min. The resulting microsomal pellet was suspended 
in 10 mM Tris-acetate buffer, pH 7.4, containing 20% glycerol and 0.1 
mM EDTA, to a protein concentration of 25 mg ml-‘, and stored at 
-20”. All centrifugations were performed at the g values determined at 
maximum radii of the rotors used. 
Solubilization and Purification of Pig Liver NADPH-Cytochrome c 
(P-450) Reductase-The microsomal suspension, 500 ml (25 mg ml-‘), 
was rapidly mixed with 100 ml of 10% Renex 690, 100 ml of 5% sodium 
deoxycholate, and 300 ml of a 20% glycerol solution which contained 
0.1 mM dithiothreitol. The mixture, which contained a final concentra- 
tion of 1% Renex 690, 0.5% sodium deoxycholate, and 12.5 mg ml-’ 
protein (a critical ratio of detergent to protein) was stirred for 30 min. 
The resulting solution was centrifuged at 105,000 x g for 60 min. Fifty 
milliliters of the supernatant were applied directly to the 2’5.ADP- 
Sepharose 4B’ as in Fig. 1 and the remainder was loaded onto a 1200.ml 
bed volume of DEAE-cellulose in a scintered glass funnel previously 
equilibrated with 25 mM Tris buffer, pH 7.7, containing 0.8% Renex 
690, 0.1% sodium deoxycholate, 0.05 mM EDTA, and 0.05 mM 
dithiothreitol. The DEAE-cellulose was washed with 4 liters of a 
similar buffer solution containing 0.12 M KCl, and the reductase was 
then eluted with 2 liters of a similar buffer solution containing 0.36 M 
KCl. The eluate was diluted 2.fold with cold distilled water and 
calcium phosphate gel (100 ml of 80 mg equivalent of dry weight ml-‘) 
was added to it. The mixture was stirred for 10 min and centrifuged at 
6,370 x g for 10 min. The reductase was eluted from the pellet by 
stirring for 60 min with 500 ml of 0.3 M potassium phosphate buffer, pH 
7.7, containing 10% glycerol, 0.2% Renex 690, and 0.05 mM dithio- 
threitol. The elution from calcium phosphate gel was repeated with a 
small volume of the latter buffer. The resulting reductase fraction was 
concentrated in an Amicon concentrator and applied to two Ultrogel 
AcA 34 columns (5.0 x 100 cm) previously equilibrated with 50 mM 
Tris buffer, pH 7.7, containing 0.2% Renex 690, 20% glycerol, 0.15 M 
KCI, 0.1 mM EDTA, and 0.1 mM dithiothreitol. The peak reductase 
fractions from Ultrogel AcA 34 columns were pooled and applied to a 
2’5.ADP-Sepharose 4B column equilibrated with 10 mM potassium 
phosphate buffer, pH 7.7, containing 0.1% Renex 690, 20% glycerol, 
0.02 mM EDTA, and 0.1 mM dithiothreitol. The column was washed 
with 200 mM potassium phosphate, pH 7.7, containing0.1% Renex 690, 
20% glycerol, 0.4 mM EDTA, and 0.1 mM dithiothreitol. No elution of 
the reductase was obtained. After washing the column again with 10 
mM potassium phosphate buffer, the reductase was eluted with a 
similar buffer solution containing 0.7 mM 2’.AMP. Detergent and 
2’-AMP were removed according to the method to be described for the 
purification of the rat reductase. The amount of reductase which can 
be bound to the 2’,5’-ADP-Sepharose 4B depends upon the degree of 
purity of the preparation applied to the column as will be illustrated in 
Figs. 1 and 2. 
Preparation of Rat Liver Microsomes-Rats were injected intraperi- 
toneally for 4 days with sodium phenobarbital at a dose of 80 mg/kg of 
body weight and starved during t.he last 24-h period. Rats were killed 
by decapitation and the livers were rapidly removed and perfused with 
50 to 100 ml of ice-cold 0.9% NaCl solution. Tissue homogenates (20%, 
w/v) were prepared in 0.25 M sucrose containing 5 mM Tris buffer plus 
0.5 mM EDTA, pH 7.5, in a Potter-Elvehjem-type homogenizer with a 
Teflon pestle at 5”. The homogenate was centrifuged at 1,900 x g for 10 
min and the supernatant was recentrifuged at 14,620 x g for 15 min. 
The resulting supernatant was centrifuged at 105,600 x g for 60 min 
and the pellet was resuspended in 0.1 M sodium pyrophosphate buffer, 
pH 7.4, and centrifuged again at 105,000 x g for 60 min. The 
microsomal pellet was resuspended to a protein concentration of 14 mg 
ml-’ in 10 mM Tris-acetate buffer, pH 7.4, containing 20% glycerol and 
0.1 mM EDTA, and stored at -80”. 
Solubilization and Purification of Rat Liver NADPH-Cytochrome c 
(P-450) Reductase-A microsomal suspension, 115 ml (about 14 mg 
ml-‘), was mixed with 20 ml of 5% sodium cholate and 20 ml of 10% 
Renex 690 and stirred for 60 min, and the solution was centrifuged at 
105,600 x g for 90 min. The supernatant fraction was app,,ed to a 
DEAE-cellulose column (Whatman DE52, 3.0 x 16 cm) previously 
equilibrated with 25 rnM Tris buffer, pH 7.7, containing 0.8% Renex 
690, 0.1% sodium cholate, 0.05 rnM EDTA, and 0.05 mM dithiothreitol. 
‘The abbreviation used is: 2’,5’-ADP-Sepharose 4B, Sepharose 
4B-bound N6-(6.aminohexyl)-adenosine 2’,5’-bisphosphate. 
The column was washed with 400 ml of equilibration buffer, and the 
NADPH-cytochrome c (P-450) reductase was then eluted with 1 liter of 
a similar solution in which the KC1 concentration was increased to 0.35 
M in a linear gradient. The active,reductase fractions were concen- 
trated in an Amicon concentrator and then applied to a 2’,5’-ADP- 
Sepharose 4B column (1.7 x 12.4 cm) previously equilibrated with 10 
mM potassium phosphate buffer, pH 7.7, containing 20% glycerol, 
0.1% Renex 690, 0.02 mM EDTA, and 0.2 mM dithiothreitol. The 
column was washed with 150 ml of 200 mM potassium phosphate 
buffer, pH 7.7, containing 20% glycerol, 0.1% Renex 690, 0.4 mM 
EDTA, and 0.2 mM dithiothreitol. No reductase activity could be 
observed. After the column was washed with 40 ml of equilibration 
buffer, the reductase was eluted with 150 ml of a similar solution in 
which the 2’.AMP concentration was increased to 2.0 mM in a linear 
gradient or eluted batchwise with 0.7 mM 2’.AMP. The reductase 
fractions were concentrated and passed through a Sephadex G-25 
column to remove unbound 2’-AMP or through an Ultrogel AcA 34 
column to remove 2’.AMP and several minor bands, which were ob- 
served on sodium dodecyl sulfate gel electrophoresis, and subsequently 
concentrated. For the titration experiment (Fig. 7), the final prepara- 
tion was passed through a small column containing 8 mg of charcoal 
and 16 mg of ‘Celite to remove traces of 2’-AMP, a method which has 
been applied to the removal of NADP+ from the reductase (8). Renex 
690 was then removed by application of the reductase to a DEAE-cellu- 
lose column which had been equilibrated with 10 mM potassium phos- 
phate buffer, pH 7.7, containing 20% glycerol. Fractions were monitored 
at 280 nm until the absorbance decreased to >0.03. The reductase 
was then eluted with 0.3 M potassium phosphate, pH 7.7, containing 
20% glycerol, and 0.1% deoxycholate. Finally, the reductase (2 to 5 ml) 
was dialyzed against 4 liters of 50 mM potassium phosphate buffer, pH 
7.7, containing 20% glycerol, 0.1 mM EDTA, and 0.1% sodium deoxy- 
cholate. 
Prlyacrylamide Disc Gel Electrophoresis in Sodium Dodecyl Sul- 
fate-Disc gels were prepared for sodium dodecyl sulfate-polyacrylam- 
ide gel electrophoresis, according to the method of Weber and Osborn 
(18), in order to determine purity of the reductase preparations. 
Samples of affinity-chromatographed reductase were applied to the 
gels and run for 4 h in potassium phosphate buffer, pH 7.2, containing 
0.1% sodium dodecyl sulfate, resulting in a single band upon electro- 
phoresis. Estimation of the molecular weight of NADPH-cytochrome c 
(P-450) reductase by sodium dodecyl-sulfate-polyacrylamide disc gel 
electrophoresis was performed by the procedure of Weber and Osborn 
(18) in the presence of the purified standard proteins: &galactosidase, 
phosphorylase a, bovine serum albumin, catalase, and aldolase. 
The Sepharose 4B-bound N6-(6.aminohexyl).adenosine 2’,5’-bis- 
phosphate was kindly supplied by Dr. Robert Bywater of Pharmacia 
Fine Chemicals prior to its introduction on the commercial market. Dr. 
Klaus Mosbach, who published the original preparation procedure (7), 
was instrumental in developing the procedure for Pharmacia and in 
directing us to this source. 
The partially purified cytochrome P-450 (specific content = 8.43 
nmol mg-‘) and dilauroylphosphatidylcholine (20 mg ml-’ in chloro- 
form) were generously supplied by Dr. Anthony Y. H. Lu of Hoffman- 
LaRoche, Inc., Nutley, N. J. Renex 690 was obtained through the 
generosity of Mr. Henry Swab of ICI, United States, Inc., Wilmington, 
Del. NADPH, NADP+, and 2’-AMP were purchased from P-L Bio- 
chemicals, Inc. FAD and FMN were purchased from Sigma Chemical 
Co. and purified by column chromatography as described previously 
(8). Cholic acid (A grade) and sodium deoxycholate (A grade) were 
purchased from Calbiochem. Ultrogel AcA 34 was obtained from LKB 
Instruments, Inc. 
Determination of Protein Concentration-Protein concentration was 
determined by the method of Lowry et al. (19) as modified by the 
procedure of Dulley and Grieve (20) for solutions containing deter- 
gents. 
Determination of NADPH-Cytochrome c Reductase Actiuity-All 
previous determinations of NADPH-cytochrome c reductase activity in 
this laboratory were performed in 0.05 M potassium phosphate buffer, 
pH 7.7, 0.1 mM EDTA at 25” (16). For purposes of comparison, 
however, with the data of Vermilion and Coon (1) and Dignam and 
Strobe1 (2). it has been necessary in presenting these data to perform 
these assays also in 0.3 M potassium phosphate, pH 7.7, 0.1 mM EDTA 
at 30”. Under these conditions, a doubling of the specific activity was 
obtained (see Table I). Therefore, it should be noted that the specific 
activity reported in Table II for liver microsomes from phenobarbital- 
pretreated rats is substantially higher (about twice) that obtained in 
most laboratories for NADPH-cytochrome c reductase activity. 
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Affinity-chromatographed NADPH-Cytochrome c (P-450) Reductase 
TABLE I 
Comparison ofspecific activities and turnover numbers ofpig and rat 
liver microsomal NADPH-cytochrome c (P-450) reductase” 
0.05 M potassium 0.3 M potassium 
phosphate, pH 7.7- phosphate, pH 7.7- 
0.1 xn~ EDTA 25” 0.1 rn~ EDTA 30” 
Specific TUIlKWt?r Specific TUrnOVer 
activity” number activity’ number 
pm01 -I firno L 
min- I mg~ I mln min- I mg~ 8 mln 
Pig liver reductase 17.8 1360 37.4 2856 
Rat liver reductase 21.9 1365 43.8 2730 
a NADPH-cytochromec reductase activities were assayed as de- 
scribed under “Experimental Procedure” under the two separate sets 
of conditions. 
b The enzyme preparations presented in this table do not represent 
the highest specific activities obtainable, but have been deliberately 
chosen to illustrate that the flavoprotein obtained by the affinity 
chromatography procedure outlined in this paper is spectrally pure. 
This is demonstrated in the turnover numbers which are calculated on 
the basis of flavin concentration in the preparations. A comparison of 
these turnover numbers with those obtained with proteolytically 
solubilized reductase in 0.05 M potassium phosphate buffer, pH 7.7,0.1 
mM EDTA at 25” (8, 11, 12) reveals that identical activities are 
obtained with the detergent-solubilized enzyme preparations. 
TABLE II 
Purification of rat liver NADPH-cytochrome c reductase by affinity 
chromatography on 2’,5’-ADP-Sepharose 4B 
The reaction mixtures contained, in a final volume of 1.0 ml, 300 
rmol of potassium phosphate buffer (pH 7.7), 0.1 rmol of NADPH, and 
0.04 rmol of cytochrome c. Assays of rat liver microsomes and 
solubilized microsomes contained 0.6 pmol of KCN in a total volume of 
1 ml. The rates of reaction were measured at 30” in 0.3 M potassium 
phosphate buffer (pH 7.7) as described under “Experimental Proce- 
dure.” This purification procedure results in spectrally pure reductase 
(42.7 /IM in total flavin) comparable to that shown in Fig. 4 (tube 2) 
with a turnover number based on flavin (1490 min. ’ at 25”; 2790 min ’ 
at 30”) equal to homogeneous enzyme Fig. 4 (tubes 3 and 4), i.e. minor 
contaminants do not interfere with activity or spectral measurements. 
Preparation Volume 
ml 
1. Microsomes 115 
2. Solubilized 191 
microsomes 
3. DEAE-cellulose 17 
column elu- 
ate 
4. 2’,5’-ADP-Sepharose 2.1 
4B column 
eluate 
5. Ultrogel AcA 34 2.3 
columns elu- 
ate 
Total Yield Total 
protein a’- 
Specific from 
tivity activity micro- somes 
vi pmoll wdl min minlmg % 
1640 762 0.46 100 
1480 762 0.51 100 
110 519 4.72 68 
6.70 366 54.6 48 
5.24 304 58.0 40 
RESULTS 
Pig liver microsomal NADPH-cytochrome c (P-450) reduc- 
tase was purified on 2’,5’-ADP-Sepharose 4B. The supernatant 
from a 100,000 x g for 60 min centrifugation of microsomes 
solubilized by Renex 690 and sodium deoxycholate (“Experi- 
mental Procedure”) was applied to the Sepharose 4B column 
(1.0 x 2.5 cm) pre-equilibrated with 10 mM potassium phos- 
phate buffer, pH 7.7, containing 20% glycerol, 0.1% Renex 690, 
0.02 mM EDTA, and 1.0 mM dithiothreitol. In Fig. 1, it can be 
seen that only about 40% of the reductase is adsorbed onto the 
column, but the adsorbed enzyme could not be eluted with 100 
mM potassium phosphate, pH 7.7, containing 20% glycerol, 
0.1% Renex, 0.2 mM EDTA, and 1.0 mM dithiothreitol (Fig. 1, 
Arrow A). After re-equilibration of the column with the original 
10 mM buffer system (Fig. 1, Arrow B), 1 mM NADH was added 
to the buffer with no effect (Fig. 1, Arrow, C). After re-equili- 
bration (Fig. 1, Arrow D), 1 mM NADPH was added (Fig. 1, 
Arrow E) resulting in elution of 76% of the bound reductase 
from the affinity column. The eluted enzyme represented a 
loo-fold purification from the microsome-bound state. 
The amount of reductase which could be bound by the 
affinity column from solubilized pig liver microsomes was % to 
% as much as could be bound in a partially purified form, i.e., 
after two chromatography steps utilizing DEAE-cellulose and 
Ultrogel AcA 34, as will be illustrated in Fig. 2. 
Affinity Chromatography of Partially Purified Pig Liver 
Microsomal NADPH-Cytochrome c (P-450) Preparation-Fig. 
2 illustrates the affinity chromatography of pig liver micro- 
somal NADPH-cytochrome c (P-450) reductase which has been 
chromatographed batch-wise on DEAE-cellulose and sub- 
jected to gel filtration on Ultrogel AcA 34. It can be seen that 
approximately 3 times the amount of reductase has been 
bound to the same column of 2’,5’-ADP-Sepharose 4B as was 
bound in the experiment of Fig. 1. The addition of 100 mM 
potassium phosphate buffer, pH 7.7 (as in Fig. l), was made as 
shown in Fig. 2, Arrow A. After re-equilibration (as in Fig. 1) 
with 10 mM potassium phosphate, pH 7.7 (Fig. 2, Arrow B), a 
linear gradient from 0 to 5 mM 2’-AMP was run in the same 
buffer as at B (Fig. 2, Arrow C). 
The choice of 2’.AMP was made on the basis of the 
knowledge that PI-AMP is a potent competitive inhibitor of 
NADPH-cytochrome c reductase activity (21) and that the 
group most crucial for the binding of NADPH to the reductase 
is most likely the 2’-phosphate group on the ribose moiety of 
the adenosine. Furthermore, it has been demonstrated (data 
not shown) that 5 mM 5’-AMP is totally ineffective in eluting 
the reductase from the affinity column and that 5’-ADP is only 
partially effective at concentrations in excess of 2.5 mM. It was 
shown in Fig. 1 that NADH is also ineffective in eluting the 
reductase. 
Determination of FAD and FMN Content of Purified Deter- 
gent-solubilized Pig Liver NADPH-Cytochrome c (P-450) 
Reductase-The FAD and FMN content of detergent-solubil- 
ized pig liver microsomal NADPH-cytochrome c (P-450) reduc- 
tase was determined by the method of Faeder and Siegel (22) 
as applied to the proteolytically solubilized, purified prepara- 
tions of the flavoprotein from rat and pig liver (8, 10) and from 
pig kidney (23). The flavin content, determined on duplicate 
samples of pig liver reductase from which 66% of the flavin was 
released, was shown to be 1 mol each of FAD and FMN, in 
agreement with the results of Vermilion and Coon (1) and 
Dignam and Strobe1 (2). 
Affinity Chromatography of Partially Purified Rat Liver 
Microsomal NADPH-Cytochrome c (P-450) Reductase-An 
aliquot containing 21.0 IU (25”) of reductase, solubilized with 
1.0% Renex 690 and 0.5% sodium cholate from rat liver 
microsomes and partially purified by gradient elution from 
DEAE-cellulose, was applied to the 2’,5’-ADP-Sepharose 4B 
column (Fig. 3) pre-equilibrated with 10 mM potassium phos- 
phate buffer as described under “Experimental Procedure” 
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5340 Affinity-chromatographed NADPH-Cytochrome c (P-450) Reductase 
TUBE NUMBER TUBE NUMBER 
FIG. 1 (left). Affinity chromatography of detergent-solubilized pig 
liver microsomes on Sepharose 4B-bound N6-(6-aminohexyl)-adeno- 
sine 2’,5’-bisphosphate. The supernatant from a 105,000 x g, 60 min 
centrifugation of pig liver microsomes, solubilized with 1.0% Renex 690 
and 0.5% sodium deoxycholate, and containing a total activity of 39.8 
IU (25’) of NADPH-cytochrome c reductase in 50 ml (specific activity 
of 0.09 IU/mg) was applied to a 2’5.ADP-Sepharose 4B column (1.0 x 
2.5 cm), equilibrated with 10 mM potassium phosphate buffer, pH 7.7, 
containing 20% glycerol, 0.1% Renex 690, 0.02 mM EDTA, and 1 rnM 
dithiothreitol. Only approximately 15.2 IU (25’) of reductase were 
adsorbed under these conditions, as can be seen by the broad, initial 
peak of reductase activity which immediately emerged from the 
column. Applications of elution buffer are indicated by the arrows: A, 
approximately 30 ml of 100 mM potassium phosphate buffer, pH 7.7, 
containing 20% glycerol, 0.1% Renex 690, 0.2 rnM EDTA, and 1 mM 
dithiothreitol; B, about 15 ml of the equilibrating buffer; C, about 12 ml 
of 1 mM NADH in the same buffer as B; D, about 15 ml of the same as 
R; E, about 12 ml of 1 mM NADPH in the equilibrating buffer. 
FIG. 2 (center). Affinity chromatography of a pig liver NADPH- 
cytochrome c reductase fraction from Ultrogel AcA 34 (Step 2) on 
Sepharose 4B-bound N’-(6.aminohexyl).adenosine 2’,5’-bisphosphate. 
Pig liver microsomalNADPH-cytochrome c reductase, solubilized 
with 1.0% Renex 690 and 0.5% sodium deoxycholate, partially purified 
by DEAE-cellulose and Ultrogel AcA 34 chromatography, and contain- 
ing a total activity of 47.1 IU (25”) in 17 ml (specific activity of 1.38 
and Fig. 1. Increasing the buffer concentration to 100 mM 
potassium phosphate buffer, pH 7.7, did not elute the reduc- 
tase, but after re-equilibration with the original buffer, 12.5 ml 
of 0.7 mM 2’-AMP eluted the reductase. In another experiment, 
not shown, 5 mM 5’.AMP was applied to the column, and no 
elution of the reductase was obtained. These data, with that 
obtained for the pig liver microsomal NADPH-cytcchrome c 
(P-450) reductase, support the conclusion that the chromatog- 
raphy is biospecific and that the 2’-phosphate group of the 
ribose attached to the adenine ring is crucial for the binding 
and subsequent elution from the 2’5.ADP-Sepharose 4B 
column. 
Typical Purification Scheme for Rat Liver Microsomal 
NADPH-Cytochrome c (P-450) Reductase-A typical purifica- 
tion scheme is shown in Table II for the rat liver microsomal 
NADPH-cytochrome c (P-450) reductase. The enzyme 
40.. 
30.. 
2 
% 
;1 2 20.. 
3 A B ( 
4 & 
IO 
0 L 
20 40 60 
TUBE NUMEt 
IU/mg) was applied to a 2’,5’-ADP-Sepharose 4B column (1.0 x 2.5 
cm), equilibrated with 10 rnM potassium phosphate, pH 7.7, containing 
20% glycerol, 0.1% Renex 690, 0.02 mM EDTA, and 0.1 mM dithio- 
threitol. Applications of elution buffer are indicated by arrows: A, ap- 
proximately 40 ml of 100 mM potassium phosphate buffer, pH 7.7, 
containing 20% glycerol, 0.1% Renex 690, 0.2 mM EDTA, and 0.1 mM 
dithiothreitol; B, about 20 ml of the equilibrating buffer; C, a linear 0 
to 5 mr.r 2’-AMP gradient in the equilibrating buffer (about 30 ml of 
equilibrating buffer in one bottle mixed equally dropwise with 30 ml of 
the same buffer containing 5 mM 2’.AMP). 
FIG. 3 (right). Affinity chromatography of a rat liver NADPH-cyto- 
chrome c reductase fraction from DEAE-cellulose (Step 1) on Sepha- 
rose 4B-bound W-(6-aminohexyl)-adenosine 2’,5’-bisphosphate. Rat 
liver microsomal NADPH-cytochrome c reductase, solubilized with 
1.0% Renex 690 and 0.5% sodium cholate, partially purified by 
gradient elution from DEAE-cellulose, and containing 21.0 IU (25”) of 
activity was applied to a 2’,5’-ADP-Sepharose 4B column (1.0 x 2.5 
cm) equilibrated with 10 mM potassium phosphate buffer, pH 7.7, con- 
taining 20% glycerol, 0.1% Renex 690, 0.02 mM EDTA, and 1 mM 
dithiothreitol. Applications of elution buffer are indicated by arrows: 
A, approximately 40 ml of 100 mM potassium phosphate buffer, pH 7.7, 
containing 20% glycerol, 0.1% Renex 690, 0.2 rnM EDTA, and 1 rnM 
dithiothreitol; B, about 15 ml of the equilibrating buffer; C, about 15 
ml of 0.7 mM 2’.AMP in the equilibrating buffer. 
obtained after Step 4 has a slightly higher specific activity than 
either the preparation of Vermilion and Coon (1) or the one of 
Dignam and Strobe1 (2), measured under identical assay 
conditions, but still exhibits minor bands on sodium dodecyl 
sulfate polyacrylamide gel electrophoresis. The enzyme is 
homogeneous after gel filtration on LKB Ultrogel ACA 34 (Step 
5), but there is little improvement in specific activity at this 
stage. 
Disc Gel Electrophoresis of Affinity-chromatographed 
Reductase-Fig. 4 shows the typical disc gel electrophoresis 
patterns on sodium dodecyl sulfate gels of affinity-chromato- 
graphed rat liver NADPH-cytochrome c (P-450) reductase 
after Step 4 (Fig. 4, tube 2) and Step 5 (Fig. 4, tubes 3 and 4) of 
the purification procedure of Table II. A comparison of the 
reductase preparations at these stages of purification shows 
that homogeneous reductase is obtained only after a final gel 
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Affinity-chromatographed NADPH-Cytochrome c (P-450) Reductase 5341 
filtration step (Fig. 4, tubes 3 and 4). Tube 3 contained 30 FL& 
and tube 4 contained 60 wg of purified reductase, and a control 
gel to which no protein was applied is shown in Fig. 4 (tube I). 
Estimation of Molecular Weight of NADPH-Cytochrome c 
(P-450) Reductase by Sodium Dodecyl Sulfate-Polyacrylamide 
Disc Gel Electrophoresis-In Fiz. 5, an experiment to estimate 
the molecular weight of rat liver NADPH-cytochrome c (P-450) 
by sodium dodecyl sulfate-polyacrylamide-disc gel electropho- 
resis by the method of Weber and Osborn (18) is shown. The 
protein standards used were P-galactosidase (M, = 130,000), 
phosphorylase a (M, = 92,500), bovine serum albumin (M, = 
68,000), catalase (M, = 57,500), and aldolase (M, = 40,000). 
The molecular weight of the reductase was estimated to be 
78,000 from the experiment of Fig. 5, a value within experimen- 
tal error of that (79,000) obtained by Vermilion and Coon (1) 
and Dignam and Strobe1 (2). The proteolytically solubilized 
reductase exhibits an estimated molecular weight of 71,000 g 
mol-’ on sodium dodecyl sulfate polyacrylamide disc gel 
electrophoresis (23). 
Recombination of Affinity-chromatographed NADPH-Cyto- 
chrome c (P-450) Reductase with Cytochrome P-450 and 
Dilauroylphosphatidylcholine to Yield Benzphetamine N- 
Demethylation-Using a preparation of cytochrome P-450, 
generously supplied by Dr. Anthony Y. H. Lu of Hoffman- 
LaRoche, Inc., the saturating amount of dilauroylphos- 
FIG. 4. Electrophoretic homogeneity of purified rat liver NADPH- 
cytochrome c (P-450) reductase. The enzyme preparations were treated 
with sodium dodecyl sulfate and mercaptoethanol and submitted to 
polyacrylamide gel electrophoresis by the method of Weber and 
Osborn with a 10% separating gel. The direction of migration was top 
to bottom. The gels were stained with Coomassie brilliant blue in a 
mixture of 454 ml of 50% methanol and 4.6 ml of glacial acetic acid, 
and destained in 7.5% acetic acid. I, no protein applied; 2, eluate from 
2’,5’-ADP-Sepharose 4B (about 55 ag); 3 and 4, eluate from Ultrogel 
AcA 34 (about 30 pg and 60 rg). 
phatidylcholine required for benzphetamine N-demethylation 
was determined at a level of cytochrome P-450 equal to 0.29 
nmol (0.035 mg of protein) and of reductase activity equal to 
0.286 pmol of reduced cytochrome c min-’ (0.0052 mg of 
protein) (cytochrome P-450 to reductase ratio of 4.4:1, data not 
shown). In a subsequent experiment, shown in Table III, the 
effects of various omissions from the complete incubation 
system are shown. The omission of reductase, cytochrome 
P-450, or benzphetamine resulted in the zero time control 
activity, but omission of NADPH gave an even lower value. 
The absolute activity values obtained are strictly comparable 
to those obtained by Lu et al. (24) under similar conditions 
?* IS- 
opGolactosidose 
L! 
~ ‘; Phosphorylase 
ze 
Gj 
NADPH-Cytochroms P-450 
7 %ovine Serum Albumin 
Reductaae 
3 
z 
6 0 Cot01aoe 
$5 
P 
;< 4 
I 
;_I\\_\; Aldolase --r-r 
04 e2 0.3 0.4 
RELATIVE MOBILITY 
FIG. 5. Estimation of the molecular weight of rat liver NADPH- 
cytochrome c (P-450) reductase by sodium dodecyl sulfate polyacryl- 
amide disc gel electrophoresis. Gels and samples were prepared as 
shown in Fig. 4. Migration of tracking dye was 10.7 cm. The marker 
proteins were fl-gaiactosidase, phosphorylase a, bovine serum albumin, 
catalase, and aldolase. Electrophoresis was carried out on three gels 
containing all proteins together and on one gel containing phosphoryl- 
ase a, NADPH-cytochrome c reductase, and bovine serum albumin, 
separately. 
TABLE III 
NADPH-dependent benzphetamine N-demethylation using 
affinity-chromatographed NADPH-cytochrome c (P-450) reductase 
and dilauroylphosphatidylcholine and cytochrome P-450 
The reaction mixture contained 150 amol of potassium phosphate 
(pH 7.4). 5 amol ofMgClz, 5 pmol of semicarbazide, 0.5 rmol of 
NADPH, 1.5 pmol of benzphetamine, rat liver NADPH-cytochrome c 
(P-450) reductase (catalyzed 0.286 Fmol of reduced cytochrome c 
min-‘; 0.0052 mg of protein), cytochrome P-450 (0.29 nmol, 0.035 mg of 
protein), and lipid (0.10 mg) in a total volume of 1.5 ml. The molar 
ratio of cytochrome P-450 to NADPH-cytochrome c (P-450) reductase 
in the reaction mixture is approximately 4:l. The reaction mixture was 
incubated at 37” for 10 min. After stopping the reaction with 0.25 ml of 
20% ZnSO, and 0.25 ml of saturated Ba(OH), and removing the 
precipitate by centrifugation (14,009 rpm for 10 min), 1.0 ml of the 
supematant was mixed with 0.4 ml of double strength Nash reagent, as 
described by Lu et al. (24). The mixture was heated at 60” for 20 min 
and the absorbance at 412 nm was recorded. For comparison with the 
rates published by Lu et al. (24), the control rate in the absence of 
benzphetamine should be subtracted. 
Conditions 
1. Complete system 
2. Minus reductase 
3. Minus cytochrome P-450 
4. Minus NADPH 
5. Minus benzphetamine 
6. Complete system at zero time 
Activity 
nnol HCHOIIO nin 
86 
20 
20 
12 
24 
22 
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5342 Affinity-chromatographed NADPH-Cytochrome c (P-450) Reductase 
0.6 
L nmoles FERRICYANIOE 
0.5 I.0 
nmolrs FERRICYANIDE / nmole FLAVIN 
\ (REDUC~I~LE) 
340 400 500 600 700 
WAVELENGTH IN nm 
FIG. 6. Aerobic titration of affinity-chromatographed pig liver deter- 6 show equilibrium absorption spectra after the addition of 6.3, 12.6, 
gent-solubilized NADPH-cytochrome c reductase with K,Fe(CN),. The 18.9, 25.2, and 34.7 nmol of K,Fe(CN)6, respectively. The total volume 
reductase purified as described under “Experimental Procedure” change was less than 5.5% and was not corrected for upon each addi- 
(31.2 run01 of total flavin) in 0.05 mM potassium phosphate buffer, pH tion. The inset shows the changes occurring at 453, 503, and 585 nm 
7.7, containing 0.1 rn~ EDTA, 20% glycerol, and 0.1% sodium deoxy- during the titration and the abscissa indicates total nanomoles of 
cholate was titrated with 2.1 mM K,Fe(CN)G in the above buffer at 25”. K,Fe(CN)s as well as the ratio of K,Fe(CN), to reducible flavin (in this 
To obtain the air-stable, half-reduced enzyme, 4 mol of NADPH/mol case, 85% based on the addition of 50-fold excess NADPH under 
of flavin were added aerobically and the flavoprotein was allowed to aerobic conditions). The specific activity of the flavoprotein used in 
reoxidize to the half-reduced state, which was confirmed by monitor- this experiment was 36 /~mol min-‘mg-’ at 30” in 0.3 M potassium 
ing at 340 nm and 453 nm. Curve 1, half-reduced enzyme; curues 2 to phosphate, pH 7.7, and a turnover number of 1360 min’ at 25”. 
FIG. 7. Aerobic titration of rat liver microsomal NADPH-cyto- 
chrome c (P-450) reductase with NADPH and KsFe(CN),. Absorbance 
changes at 453 (O), 500 (O), and 585 (0) nm are plotted against either 
NADPH (A) or K,Fe(CN), (B) added. Rat liver microsomal NADPH- 
cytochrome c (P-450) reductase was in 0.05 M potassium phosphate 
(pH 7.7) containing 0.1% sodium deoxycholate and 20% glycerol to a 
final concentration of 7.52 pM in a total volume of 3 ml and titrated 
with NADPH and K,Fe(CN), under aerobic conditions at 25”. 
Experiments A and B used the same enzyme preparation but were 
carried out separately. The enzyme preparation was 85% reducible as 
determined by the addition of 50 ma1 of NADPH/mol of flak. Total 
volume change was less than 3.5% and was not corrected for upon 
addition. 
(Table II). The residual activity (about 20%) which is mea- 
sured in the absence of reductase, or cytochrome P-450, or 
benzphetamine was also obtained in the absence of lipid and 
was not subtracted from the rate obtained with the complete 
incubation system. 
Aerobic Back Titration of Air-stable Half-reduced Pig Liver 
NADPH-Cytochrome c (P-450) Reductase Purified by Affinity 
Chromatography-In order to ascertain the electron transfer 
properties of the detergent-solubilized, affinity-chromato- 
graphed reductase, the experiment shown in Fig. 6 was 
performed. The flavoprotein was reduced with excess NADPH 
(4 mol of NADPH/mol of total flavin, i.e., 126 nmol of 
NADPH) aerobically and allowed to oxidize in air until there 
was no further change at 340 nm or 453 nm, indicating 
complete oxidation of the NADPH and no further oxidation of 
flavin, respectively. Potassium ferricyanide (2.1 mM) was 
added in aliquots resulting in the various equilibrium absorp- 
tion spectra shown in Fig. 6. The changes in absorbance at 453, 
503, and 585 nm are shown in the inset, and the abscissa 
indicates both total nanomoles of potassium ferricyanide 
added and the ratio of the oxidant to reducible flavin. The 
results show that 1 mol of potassium ferricyanide/mol of 
enzymically reducible flavin is required to reoxidize the 
flavoprotein to its fully oxidized state, indicating that 2 
electrons remain in the stable, half-reduced form of the 
reductase (containing 1 mol each of FAD and FMN) in 
agreement with previous results of Masters et al. (8, 10) and 
Kamin (9). The previous titration results reported by Masters 
et al. (8, 10) and Kamin (9) were obtained with the proteolyti- 
tally prepared reductase. The present data have been obtained 
with detergent-solubilized reductase, purified by affinity chro- 
matography techniques to a specific activity with cytochrome c 
as electron acceptor of 36 wmol min-‘mg-’ as measured in 0.3 
M potassium phosphate, pH 7.7, at 30”, a specific activity 
comparable to that obtained by Vermilion and Coon (1) and 
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Affinity-chromatographed NADPH-Cytochrome c (P-450) Reductase 5343 
Dignam and Strobe1 (2) for the purified rat liver reductase 
preparations. 
Aerobic Titration of Rat Liver Microsomal NADPH-Cyto- 
chrome c (P-450) Reductase with NADPH and Potassium 
Ferricyanide-Fig. 7 illustrates a typical titration experiment 
in which oxidized reductase was titrated aerobically with 
aliquots of NADPH to the air-stable, reduced form (Fig. 7A), 
at which point the curve breaks and no further reduction of 
flavin is recorded at 453 nm. It is also seen that the isosbestic 
point at 500 nm for oxidized and half-reduced reductase is 
unaffected by the addition of NADPH. The longer wavelength 
absorption increases until maximal formation of the air-stable, 
half-reduced spectrum is obtained. When the amount of 
NADPH added equals 0.5 mol/mol of reducible flavin, the 
titration curves at both 453 and 585 nm are seen to break. In a 
separate experiment on the same enzyme preparation, excess 
NADPH (1.5 mol/mol of flavin) was added and the reductase 
was allowed to reoxidize to the air-stable, reduced form, at 
which time aliquots of K,Fe(CN), were added to reoxidize the 
enzyme to its fully oxidized state (Fig. 7B). Again, it can be 
seen that 1.0 mol of K,Fe(CN),/mol of reducible flavin is 
required to reoxidize the flavoprotein from the air-stable 
reduced state to its fully oxidized state, confirming the results 
obtained in this paper (Fig. 6) on detergent-solubilized pig 
liver microsomal NADPH-cytochrome c reductase and earlier 
data on the proteolytically solubilized enzymes from pig and 
rat liver (8-10). These data unequivocally demonstrate that 
the spectral species which is obtained aerobically upon reduc- 
tion with NADPH to an air-stable, partially reduced state and 
which results from reoxidation of the fully reduced enzyme 
with various electron acceptors (11, 12) does, indeed, contain 2 
electron equivalents. 
DISCUSSION 
The foregoing paper has described purification procedures 
for pig and rat liver microsomalNADPH-cytochrome c (P-450) 
reductases utilizing biospecific affinity chromatography on 
Sepharose 4B-bound NO-(6.aminohexyl)-adenosine 2’,5’-bis- 
phosphate (7). The procedures involve either prior batchwise 
chromatography on DEAE-cellulose plus gel filtration on 
Ultrogel AcA 34 for pig liver microsomal reductase or DEAE- 
cellulose column chromatography only for rat liver microsomal 
reductase. Both procedures produce spectrally pure NADPH- 
cytochrome c (P-450) reductase (such as that shown in Fig. 6) 
with a turnover number equal to that obtained with homogene- 
ous enzyme. With a subsequent chromatography step using gel 
filtration on Ultrogel AcA 34, it is possible to obtain homogene- 
ous flavoprotein as verified in Fig. 4 (tubes 3 and 4). 
Electrophoresis on sodium dodecyl sulfate gels reveals minor 
contaminants with rat liver microsomal reductase purified by 
DEAE-cellulose column chromatography and subsequent af- 
finity chromatography on 2’,5’-ADP-Sepharose 4B as shown in 
Fig. 4 (tube 2), but final gel filtration on LKB AcA 34 yields a 
preparation exhibiting a single band on disc gel electrophoresis 
(Fig. 4, tubes 3 and 4). The yields of reductase vary between 30 
and 55%, depending on the purity of enzyme desired. It should 
be noted that the purification procedure through Step 4 in 
Table II is adequate for the production of spectrally pure re- 
ductase capable of reconstituting oxidative demethylation of 
drugs with partially purified cytochrome P-450 and dilauroyl- 
phosphatidylcholine (Table III). This preparation is also ade- 
quate for spectral titration studies, i.e., the turnover number, 
based on flavin, is equal to that obtained with any homogene- 
ous preparation. The molecular weight, estimated by the 
procedure of Weber and Osborn (18) on sodium dodecyl sulfate 
gel electrophoresis in the presence of standard proteins of 
known subunit molecular weight, was estimated to be 78,000 g 
mol-’ (Fig. 5). 
Aerobic titration of pig liver microsomal reductase from the 
air-stable, reduced form to the fully oxidized form with 
potassium ferricyanide resulted in a stoichiometry of 1 mol of 
ferricyanide/mol of reducible flavin (Fig. 6). In addition, 
aerobic titration of the fully oxidized rat liver reductase to the 
air-stable, reduced form with NADPH and back titration to 
the fully oxidized form with potassium ferricyanide confirm 
that 2 electron equivalents exist in this aerobically stable 
intermediate (Fig. 7). These results are in complete agreement 
with previous results of Masters et al. (8, 10) obtained with 
proteolytically solubilized reductase. It is essential to establish 
the mechanism of NADPH-cytochrome c (P-450) reductase 
purified from detergent-solubilized microsomes, since it is 
capable of recombining with preparations of cytochrome P-450 
and dilauroylphosphatidylcholine to oxidatively demethylate 
drugs, a capacity which the proteolytically solubilized reduc- 
tase did not possess. Indeed, it is cytochrome P-450 (not 
cytochrome c, potassium ferricyanide, or 2,6-dichlorophenolin- 
dophenol) which is the terminal acceptor of electrons donated 
through this flavoprotein (25). 
The procedures outlined in this paper represent the first 
successful application of biospecific affinity chromatography 
to any microsomal electron transport component. The 2’,5’- 
ADP-Sepharose-4B columns can be utilized repeatedly with- 
out loss of binding capacity. The affinity chromatography 
medium is stable in the presence of detergents, either nonionic 
or ionic. Since this improved, high yield procedure produces 
consistently pure enzyme preparations, we plan to utilize this 
technique for the production of the large quantities of flavo- 
protein required for reconstitution experiments involving cyto- 
chrome P-450, EPR titration studies, and spectrophotometric 
and fluorimetric studies on the reactivation of the enzyme with 
various flavins. 
Acknowledgments-We would like to thank Dr. Klaus Mos- 
bath, Lunds Universitet, Sweden, for suggesting the use of the 
2’,5’-ADP-Sepharose-4B, for the instigation of whose develop- 
ment for commercial use he was primarily responsible. We are 
also grateful to Dr. Robert Bywater of Pharmacia Fine Chemi- 
cals, Uppsala, Sweden, for his cooperation in making this 
chromatography medium available to us before its introduc- 
tion on the commercial market. We owe a special note of 
thanks to Drs. Russell A. Prough and Julian A. Peterson for 
many critical discussions and helpful suggestions. 
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Y Yasukochi and B S Masters
P-450) reductase purified by biospecific affinity chromatography.
Some properties of a detergent-solubilized NADPH-cytochrome c(cytochrome
1976, 251:5337-5344.J. Biol. Chem. 
 
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