Enzymatic Assay of Proteinase K (EC 3.4.21.64)

Description

This procedure may be used for determination of Proteinase K activity using hemoglobin as the substrate. It is not used to assay the activity of immobilized Proteinase K products, such as Catalog Numbers P9290 or 82452.

The spectrophotometric stop rate determination (A750, Light path = 1 cm) is based on the following reaction::

Unit Definition: One unit of Proteinase K will hydrolyze urea-denatured hemoglobin to produce color equivalent to 1.0 µmole of tyrosine per minute at pH 7.5 at 37 °C (color by Folin & Ciocalteu's Phenol Reagent).

Reagents and Equipment Required

Potassium phosphate, monobasic (Catalog Number P5379)

Hemoglobin from bovine blood (Catalog Number H2625)

1 M NaOH solution

Urea (Catalog Number U1250)

Calcium chloride, dihydrate (Catalog Number C3881)

6.1 N Trichloroacetic acid solution (Catalog Number T0699)

2.0 N Folin & Ciocalteu's Phenol Reagent (Catalog Number F9252)

L-Tyrosine (Catalog Number T3754)

1.0 M Hydrochloric Acid

 

Precautions

Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

 

Preparation Instructions

Use ultrapure water (≥18 MΩxcm resistivity at 25 °C) for the preparation of reagents.

Buffer (1.0 M Potassium Phosphate Buffer, pH 7.5 at 37 °C) – Prepare 136 mg/ml solution of Potassium phosphate, monobasic (Catalog Number P5379) in ultrapure water. Adjust the pH to 7.5 at 37 °C with 1 M to 10 M KOH.

Substrate Solution [100 mM Potassium Phosphate, pH 7.5 at 37 °C with 2.0% (w/v) Hemoglobin and 6 M Urea] – To prepare 100 ml of Substrate Solution, add 2.0 g of Hemoglobin from bovine blood (Catalog Number H2625) to ~40 ml of ultrapure water. Allow hemoglobin to dissolve by mixing at 37 °C for 30 minutes. Add 8.0 ml of 1 M NaOH solution and mix for an additional 20 minutes at 37 °C. Add 36.0 g of Urea (Catalog Number U1250) and continue mixing at 37 °C for 60 minutes. Add 10 ml of Buffer and adjust the pH to 7.5 at 37 °C with 1 M to 5 M HCl. Bring the final volume to 100 ml with ultrapure water.

Enzyme Diluent (20 mM CaCl2 Solution) – Prepare 2.95 mg/ml solution of Calcium chloride, dihydrate (Catalog Number C3881) in ultrapure water.

TCA Solution (305 mM [5% (w/v)] Trichloroacetic Acid) – Dilute 6.1 N [~100% (w/v)] Trichloroacetic acid solution (Catalog Number T0699) 20-fold with ultrapure water.

0.5 M NaOH Solution – Dilute 1.0 M Sodium hydroxide solution 2‑fold with ultrapure water.

F&C Reagent (1 N Folin & Ciocalteu's Phenol Reagent) – Dilute 2.0 N Folin & Ciocalteu's Phenol Reagent (Catalog Number F9252) 2-fold with ultrapure water.

Tyr Standard Solution (1.1 mM L-Tyrosine standard solution) – Weigh 20 mg of L-Tyrosine (Catalog Number T3754) on a microbalance and transfer to a 100 ml Class A volumetric flask. Add ~80 ml of ultrapure water. Heat gently to 70–80 °C (do not boil) until the Tyrosine dissolves. Cool to room temperature. Bring to volumetric mark with ultrapure water.

Note: Tyr Standard Solution is stable for 6 months. Store in a refrigerator.

200 mM HCl Solution – Dilute 1.0 M Hydrochloric Acid 5‑fold with ultrapure water.

Enzyme Solution (Proteinase K) – Immediately before use, prepare a solution containing 0.075–0.175 unit/ml of Proteinase K in cold (2–8 °C) 20 mM CaCl2 Solution.

Procedure

1. Pipette the Substrate into suitable vials.

Reagent Test 1
(ml)
Test 2
(ml)
Test 3
(ml)
Test Blank
(ml)
Substrate 2.50 2.50 2.50 2.50

 

2. Equilibrate to 37 °C for ~10 minutes and then add:
 

Reagent Test 1
(ml)
Test 2 (ml) Test 3 (ml) Test Blank
(ml)
TCA Solution
Enzyme Solution 0.50 0.50 0.50

 

3. Mix by swirling, incubate at 37 °C for exactly 10 minutes, and then add:
 

Reagent Test 1
(ml)
Test 2 (ml) Test 3
(ml)
Test Blank
(ml)
TCA Solution 5.00 5.00 5.00 5.00
Enzyme Solution 0.50


4.     Mix by swirling and incubate at room temperature for 20 minutes.

5.     Clarify each solution by filtering through a 0.45 µm filter.

6.     Add the following in suitable vials:
 

Reagent Test 1
(ml)
Test 2
(ml)
Test 3
(ml)
Test Blank
(ml)
Filtrate from step 5 2.50 2.50 2.50 2.50
0.5 M NaOH Solution 5.00 5.00 5.00 5.00

 

7.     Cap the vials and mix each Test and the Test blank thoroughly by swirling.

8.     Prepare a series of standards by pipetting (in milliliters) the following reagents into suitable vials:
 

Reagent Std 1 Std 2 Std 3 Std 4 Std 5 Std Blank
Tyr Standard Solution 0.05 0.10 0.30 0.50 0.70
200 mM HCl Solution 2.45 2.40 2.20 2.00 1.80 2.50
0.5 M NaOH Solution 5.00 5.00 5.00 5.00 5.00 5.00

Note: Standard volumes may be modified or added as needed.

9. Mix each Standard and the Standard Blank thoroughly by swirling.

10.  Add 1.50 ml of F&C Reagent to all tests, standards, and blanks. Cap vials, immediately mix thoroughly by swirling or vortexing, and incubate at room temperature for 30 minutes.

11.  Blank a suitable thermostatted spectrophotometer versus air. Transfer each solution into a suitable cuvette and measure the absorbance at 750 nm.

Note: If solutions are hazy, filter through a 0.45 µm filter immediately prior to measuring the absorbance.

 

Results

Calculations

1. Standard Curve

For each standard, calculate ΔA750 Standard:

ΔA750 Standard = A750 Standard – A750 Standard Blank

Plot a standard curve of the ΔA750 Standard for the standards versus the µmoles of Tyrosine using linear regression. The R-squared value must be ≥0.99 for the results to be valid.

 

2. Test Determination

For each test, calculate ΔA750 Test:

ΔA750 Test = A750 Test – A750 Test Blank

From the Standard Curve, determine the µmoles of Tyrosine equivalents released for the Test..

Units/ml enzyme = (µmole of Tyrosine equivalents released) (8.0) (df)
(0.5) (10) (2.5)

where:

8.0 = volume (ml) of total stopped reaction

df = dilution factor

0.50 = ml of Enzyme Solution added

10 = assay incubation time (minutes)

2.5 = volume (ml) of filtrate used in the colorimetric determination

3.

Units/mg solid = unit/ml enzyme
mg solid/ml enzyme

Materials

     

 Reference

  1. Folin, O., and Ciocalteu, V., J. Biol. Chem., 73, 627-650 (1927).

 

03/17-1

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