Enzymatic Assay of Proteinase K with Hemoglobin Substrate

Proteinase K (EC 3.4.21.64) Activity Assay Description

This procedure is for determining Proteinase K (EC 3.4.21.64) activity using hemoglobin as the substrate.

Note: This assay Is not for measuring the activity of immobilized Proteinase K products, such as product 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 µmol of tyrosine per minute at pH 7.5 at 37 °C (color by Folin & Ciocalteu's Phenol Reagent).

Proteinase K Assay Reagents Required

• Potassium phosphate, monobasic (P5379)
• Hemoglobin from bovine blood (H2625)
• 1 M NaOH solution
• Urea (U1250)
• Calcium chloride, dihydrate (C3881)
• 6.1 N Trichloroacetic acid solution (T0699)
• 2.0 N Folin & Ciocalteu's Phenol Reagent (F9252)
• L-Tyrosine (T3754)
• 1.0 M Hydrochloric Acid (H9892)

Proteinase K Assay Reagent Preparation

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 (P5379) in ultrapure water. Adjust the pH to 7.5 at 37 °C with 1 M to 10 M KOH.

Substrate [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 (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 (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. Adjust 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 (C3881) in ultrapure water.

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

0.5 M NaOH Solution – Dilute 1.0 M sodium hydroxide solution two‑fold with ultrapure water.

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

Tyr Standard Solution (1.1 mM L-Tyrosine standard solution) – Weigh 20 mg of L-tyrosine (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. Adjust to the 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 (H9892) five‑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.

Proteinase K Assay Procedure

1. Pipette the Substrate into suitable vials.

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

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

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:

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:

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 before measuring the absorbance.

Calculate Assay 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 micromoles 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 micromoles of tyrosine equivalents released for the Test.

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.