This procedure is for determining Proteinase K (EC 184.108.40.206) activity using hemoglobin as the substrate.
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).
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 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.
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.
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.
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