Enzymatic Assay of Hyaluronidase (3 2 1 35)

Prévia do material em texto

27/07/2021 Enzymatic Assay of Hyaluronidase (3.2.1.35)
https://www.sigmaaldrich.com/BR/en/technical-documents/protocol/protein-biology/enzyme-activity-assays/enzymatic-assay-of-hyaluronidase 1/5
Enzymatic Assay of Hyaluronidase (3.2.1.35)
Enzymatic Assay of Hyaluronidase (3.2.1.35)
DESCRIPTION
This procedure is a turbidimetric determination (% Transmittance at 600 nm, Light path = 1 cm) of
Hyaluronidase activity. It is based on the following reaction:
Hyaluronidase
Hyaluronic acid ––––––––––––> Di and monosaccharides + smaller hyaluronic acid fragments
Unit De�nition: One unit of Hyaluronidase activity will cause a change in A of 0.330 per
minute at pH 5.35 at 37 °C in a 2.0 ml reaction mixture (45 minute assay).
REAGENTS AND EQUIPMENT REQUIRED
5.0 M Sodium phosphate monobasic solution (Catalog Number 74092)
Hyaluronic acid (Catalog Number H7630 or H5388)
0.5 M Sodium phosphate dibasic solution (Catalog Number 94046)
5.0 M Sodium chloride solution (Catalog Number S6546)
Bovine Serum Albumin (Catalog Number A4503)
Sodium acetate (Catalog Number S8625)
Acetic acid (Catalog Number 695092)
Cuvettes
Precautions – Please consult the Safety Data Sheet for information regarding hazards and safe
handling practices.
PREPARATION INSTRUCTIONS
Use ultrapure water (≥18 MΩ×cm resistivity at 25 °C) for the preparation of reagents.
Phosphate Buffer (300 mM Sodium Phosphate, pH 5.35 at 37 °C) – Prepare a 1:16.7 dilution using
5.0 M Sodium phosphate monobasic solution (Catalog Number 74092) in ultrapure water. Adjust
the pH to 5.35 at 37 °C with 1 M NaOH or 1 M HCl.
Type in Product Names, Product Numbers, or CAS Numbers to see suggestions.
600
https://www.sigmaaldrich.com/BR/en/technical-documents/protocol/protein-biology/enzyme-activity-assays/enzymatic-assay-of-hyaluronidase
https://www.sigmaaldrich.com/BR/en
27/07/2021 Enzymatic Assay of Hyaluronidase (3.2.1.35)
https://www.sigmaaldrich.com/BR/en/technical-documents/protocol/protein-biology/enzyme-activity-assays/enzymatic-assay-of-hyaluronidase 2/5
Hyaluronic Acid Solution [0.03% (w/v)]
1. Prepare a 0.3 mg/mL solution in Phosphate Buffer using hyaluronic acid (Catalog Number
H7630 or H5388).
2. Heat the solution to 90–95 °C with stirring until the hyaluronic acid is dissolved. Fifteen to
thirty minutes of heating and stirring is required for this material to completely dissolve. Do
NOT boil.
3. After dissolution, allow the solution to cool to 37 °C in a water bath. Adjust the solution to
original volume using ultrapure water. Adjust the pH to 5.35 at 37 °C with 1 M NaOH or 1 M
HCl.
Enzyme Diluent [20 mM Sodium Phosphate with 77 mM Sodium Chloride and 0.01% (w/v)
Bovine Serum Albumin, pH 7.0 at 37 °C] – To prepare 200 mL:
1. Add 8.46 mL of 0.2 M Sodium phosphate monobasic (prepared using Catalog Number 74092 in
ultrapure water).
2. Add 11.54 mL of 0.2 M Sodium phosphate dibasic (prepared using Catalog Number 94046 in
ultrapure water.
3. Add 3.08 mL of 5.0 M Sodium chloride solution (Catalog Number S6546).
4. Add 20 mg of Bovine Serum Albumin (Catalog Number A4503).
5. Add ultrapure water to make up the �nal volume.
6. Adjust the pH to 7.0 at 37 °C with 1 M NaOH or 1 M HCl.
Acidic Albumin Solution [24 mM Sodium Acetate, 79 mM Acetic Acid with 0.1% (w/v) Bovine
Serum Albumin, pH 3.75 at 25 °C] – To prepare 150 mL:
1. Add 3.6 mL of stock 1.0 M Sodium Acetate, pH 4.8, solution (prepared using Catalog Number
S8625).
2. Add 0.68 mL of Acetic Acid (Catalog Number 695092).
3. Add 150 mg of Bovine Serum Albumin (Catalog Number A4503).
4. Add ultrapure water to make up the �nal volume.
5. Adjust the pH to 3.75 at 25 °C with 5 M HCl.
Enzyme Solution (Hyaluronidase) – Immediately before use, prepare a 1,000 units/mL enzyme
stock solution in cold Enzyme Diluent. Dilute the enzyme stock solution with cold Enzyme Diluent
to obtain a working solution of ~6 units/mL
PROCEDURE
Final Assay Concentrations – In a 2.0 mL reaction mix the �nal concentrations are 0.015% (w/v)
hyaluronic acid, 150 mM sodium phosphate, and 2‑5 units of hyaluronidase. 
Note: Stagger all Tests and Blank by at least 30 seconds such that there is suf�cient time to mix
each Test and Blank by inversion before incubating at room temperature.
1. Pipette the following reagents into suitable containers:
27/07/2021 Enzymatic Assay of Hyaluronidase (3.2.1.35)
https://www.sigmaaldrich.com/BR/en/technical-documents/protocol/protein-biology/enzyme-activity-assays/enzymatic-assay-of-hyaluronidase 3/5
Reagent
Test 1
(mL)
Test 2
(mL)
Test 3
(mL)
Test 4
(mL)
Test 5
(mL)
Test 6
(mL)
Blank
(mL)
Enzyme
Solution
0.750 0.665 0.585 0.500 0.415 0.335 0.00
Enzyme
Diluent
0.250 0.335 0.415 0.500 0.585 0.665 1.00
2. Mix by swirling and equilibrate to 37 °C for 10 minutes. Then add:
Reagent
Test
1
(mL)
Test
2
(mL)
Test
3
(mL)
Test
4
(mL)
Test
5
(mL)
Test
6
(mL)
Blank
(mL)
Hyaluronic
Acid Solution
1.00 1.00 1.00 1.00 1.00 1.00 1.00
3. Immediately mix by swirling and incubate at 37 °C for exactly 45 minutes.
4. After 45 minutes, transfer 0.5 mL of each Test and Blank into suitable cuvettes containing 2.5
mL of Acidic Albumin 
Solution and mix immediately by inversion.
5. Allow each cuvette to stand for 10 minutes at room temperature. 
Note: Results will change signi�cantly if incubation time is not exactly 10 minutes.
6. Determine the % transmittance at 600 nm.
Zero the spectrophotometer against the Blank. Read the % transmittance for the Tests and the
Blank. 
Note: When batching samples together, blank the spectrophotometer against the respective
Blank solutions prepared.
The uncorrected % transmittance for each Test must be between 130–170%. A minimum of
three valid values for each Test is required to calculate the activity. 
Note: The concentration of the Enzyme Solution can be adjusted to achieve results within the
valid % transmittance range.
RESULTS
Calculations
27/07/2021 Enzymatic Assay of Hyaluronidase (3.2.1.35)
https://www.sigmaaldrich.com/BR/en/technical-documents/protocol/protein-biology/enzyme-activity-assays/enzymatic-assay-of-hyaluronidase 4/5
1.
(%T Test – %T Blank) (df) 
Units/mL enzyme = ----------------------------------------------- 
(14.84) (ml of enzyme solution)
where: 
df = dilution factor of enzyme 
14.84 = Sigma-Aldrich Determined Extinction Coef�cient 
mL of enzyme solution = Volume of enzyme solution used in reaction
2.
Materials
Product Number Product Description SDS
A4503
Bovine Serum Albumin
cold ethanol fraction, pH 5.2, ≥96%
H7630
Hyaluronic acid sodium salt from bovine vitreous
humor
H5388
Hyaluronic acid sodium salt from rooster comb
avian glycosaminoglycan polysaccharide
H3506
Hyaluronidase from bovine testes
Type I-S, lyophilized powder, 400-1000 units/mg solid
H3884
Hyaluronidase from bovine testes
Type IV-S, lyophilized powder (essentially salt-free),
750-3000 units/mg solid
H4272
Hyaluronidase from bovine testes
Type IV-S, powder, suitable for mouse embryo cell
culture, 750-3000 units/mg solid
H3757
Hyaluronidase from bovine testes
Type VIII, lyophilized powder, 300-1,000 U/mg
H3631
Hyaluronidase from bovine testes
Type VI-S, lyophilized powder, 3,000-15,000 units/mg
solid
Hyaluronidase from sheep testes
https://www.sigmaaldrich.com/BR/en/product/sigma/a4503?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/a4503?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/h7630?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/h7630?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/h5388?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/h5388?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/h3506?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/h3506?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/h3884?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/h3884?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/h4272?context=producthttps://www.sigmaaldrich.com/BR/en/product/sigma/h4272?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/h3757?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/h3757?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/h3631?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/h3631?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/h6254?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/h6254?context=product
27/07/2021 Enzymatic Assay of Hyaluronidase (3.2.1.35)
https://www.sigmaaldrich.com/BR/en/technical-documents/protocol/protein-biology/enzyme-activity-assays/enzymatic-assay-of-hyaluronidase 5/5
Product Number Product Description SDS
H6254
ya u o dase o s eep testes
Type V, lyophilized powder, ≥1,500 units/mg solid
H2126
Hyaluronidase from sheep testes
Type II, lyophilized powder, ≥300 units/mg solid
H1136 Hyaluronidase from Streptomyces hyalurolyticus
94046
Sodium phosphate dibasic solution
BioUltra, 0.5 M in H O
74092
Sodium phosphate monobasic solution
BioUltra, 5 M in H O
RELATED ARTICLES
MTT Assay Protocol for Cell Viability and Proliferation
Protocol Guide: WST-1 Assay for Cell Proliferation and Viability
Enzymatic Reactions in Ionic Liquids
Enzymatic Food Analysis
Enzymatic Assay of Malic Dehydrogenase (EC 1.1.1.37)
Enzymatic Method for Determining Alanine (Alanine Assay)
Alkaline Phosphatase (AP) Protocol
Assay Procedure for Glycerol Dehydrogenase
RELATED PRODUCT CATEGORIES
Carbohydrates
Albumins and Transport Proteins
Salts
Acids
Tissue Dissociation
Mouse Embryo Media
© 2021 Merck KGaA, Darmstadt, Germany and/or its af�liates. All Rights Reserved.
Reproduction of any materials from the site is strictly forbidden without permission.
Site Use Terms | Privacy Policy | General Terms and Conditions of Sale | Copyright Consent
2
2
https://www.sigmaaldrich.com/BR/en/product/sigma/h6254?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/h6254?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/h2126?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/h2126?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/h1136?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/h1136?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/94046?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/94046?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/74092?context=product
https://www.sigmaaldrich.com/BR/en/product/sigma/74092?context=product
https://www.sigmaaldrich.com/BR/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/cell-counting-and-health-analysis/cell-proliferation-kit-i-mtt
https://www.sigmaaldrich.com/BR/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/cell-counting-and-health-analysis/cell-proliferation-reagent-wst-1
https://www.sigmaaldrich.com/BR/en/technical-documents/protocol/chemistry-and-synthesis/reaction-design-and-optimization/enzymatic-reactions
https://www.sigmaaldrich.com/BR/en/technical-documents/protocol/food-and-beverage-testing-and-manufacturing/chemical-analysis-for-food-and-beverage/enzymatic-food-analysis
https://www.sigmaaldrich.com/BR/en/technical-documents/protocol/protein-biology/enzyme-activity-assays/_enzymatic-assay-of-malic-dehydrogenase
https://www.sigmaaldrich.com/BR/en/technical-documents/protocol/protein-biology/enzyme-activity-assays/alanine-assay-kit-mak001
https://www.sigmaaldrich.com/BR/en/technical-documents/protocol/protein-biology/enzyme-activity-assays/alkaline-phosphatase-ap
https://www.sigmaaldrich.com/BR/en/technical-documents/protocol/protein-biology/enzyme-activity-assays/assay-procedure-for-glycerol-dehydrogenase
https://www.sigmaaldrich.com/BR/en/products/chemistry-and-biochemicals/biochemicals/carbohydrates
https://www.sigmaaldrich.com/BR/en/products/cell-culture-and-analysis/cell-culture-supplements-and-reagents/albumins-and-transport-proteins
https://www.sigmaaldrich.com/BR/en/products/chemistry-and-biochemicals/lab-chemicals/salts
https://www.sigmaaldrich.com/BR/en/products/chemistry-and-biochemicals/lab-chemicals/acids
https://www.sigmaaldrich.com/BR/en/products/protein-biology/proteins-and-enzymes/tissue-dissociation
https://www.sigmaaldrich.com/BR/en/products/cell-culture-and-analysis/cell-lines-and-specialty-cell-culture/mouse-embryo-media
https://www.sigmaaldrich.com/BR/en/life-science/legal/site-use-terms
https://www.sigmaaldrich.com/BR/en/life-science/legal/privacy-statement
https://www.sigmaaldrich.com/BR/en/life-science/legal/terms-and-conditions
https://www.sigmaaldrich.com/BR/en/life-science/legal/copyright-consent