Enzymatic Assay of Proteinase K with Hemoglobin Substrate

1. Objective

The objectiveis to standardize a procedure for the enzymatic determination of Proteinase K using Hemoglobin as the substrate.

2. Scope

2.1 The scope of this procedure is all Proteinase K products that have a specification for Proteinase K activity using Hemoglobin as the substrate.

2.2 This procedure is not to be used to assay proteinase K-acrylic beads, Sigma-Aldrich Product Number P0803, and proteinase K-agarose, Sigma-Aldrich Product P9290.

3. Definitions

3.1 Purified Water - water from a deionizing system, resistivity > or = 18MΩ•cm @ 25ºC

3.2 Unit Definition - One unit will hydrolyze 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).

4. Discussion

Hemoglobin + H2O    ProteinaseK   > TCA soluble peptides and Amino Acids

5. Responsibilities

It is the responsibility of all trained Analytical Services personnel to follow this protocol as written.

6. Safety

Refer to Material Safety Data Sheets (MSDS) for hazards and appropriate handling precautions.

7. Procedure

7.1 CONDITIONS:
T = 37°C, pH = 7.5, A750nm, Light path = 1 cm

7.2 METHOD:
Spectrophotometric Stop Reaction

7.3 REAGENTS:

7.3.1    1.0 M Potassium Phosphate Buffer, pH 7.5 at 37°C (Buffer)
Prepare a 136 mg/ml solution in purified water using Potassium Phosphate, Monobasic, Anhydrous, Sigma-Aldrich Product Number P5379. Adjust to pH 7.5 at 37°C with 1 M KOH.

7.3.2    2.0% (w/v) Hemoglobin, 6 M Urea, 100 mM Potassium Phosphate, pH 7.5 at 37°C. (Hem)

7.3.2.1    Prepare 100 ml by dissolving 2.0 g of Hemoglobin, Sigma-Aldrich Product Number H2625, in approximately 40 ml of purified water.

7.3.2.2    Allow Hemoglobin to stir for 30 minutes at 37°C.

7.3.2.3    Add 8.0 ml of 1 N NaOH, Sigma-Aldrich Product Number S2567 and stir for 20 minutes at 37°C.

7.3.2.4    Add 36.0 g of Urea, Sigma-Aldrich Product Number U1250, and equilibrate to 37°C. Stir for 60 minutes at 37°C.

7.3.2.5    Add 10 ml of reagent 7.3.1 (Buffer). Adjust the pH to 7.5 at 37°C with 1 N HCl.

7.3.2.6    Adjust the volume to 100 ml with purified water.

7.3.3    20 mM Calcium Chloride (CaCl2)
Prepare a 2.95 mg/ml solution in purified water using Calcium Chloride, Sigma-Aldrich Product Number C3881.

7.3.4    305 mM Trichloroacetic Acid Reagent (TCA)
Dilute 20 ml of Trichloroacetic Acid, 6.1 N, approximately 100% (w/v), Sigma-Aldrich Product Number T0699, to 400 ml with purified water.

7.3.5    500 mM Sodium Hydroxide (NaOH)
Prepare 0.5 ml/ml solution in purified water using Sigma-Aldrich Product Number S2567.

7.3.6   1 N Folin & Ciocalteu's Phenol Reagent (F-C)
Prepare a 0.5 ml/ml solution in purified water using Folin & Ciocalteu's phenol reagent, Sigma-Aldrich Product Number F9252.

7.3.7   1.1 mM L-Tyrosine Standard (Std Soln)
Prepare 100 ml in purified water using L-tyrosine, Sigma-Aldrich Product Number T3754.

7.3.7.1    Weigh on a microbalance, using Class A volumetric glassware.

7.3.7.2    Heat gently (do not boil) to 70-80°C until the tyrosine dissolves, and cool to room temperature.

7.3.8    200 mM HCl (HCl)
Prepare a 1:5 solution using HCl , Sigma-Aldrich Product Number H3162.

7.3.9    Proteinase K Enzyme Solution (Pro K)
Immediately before use, prepare a solution containing 0.075 - 0.175 unit/ml of proteinase K in cold reagent 7.3.3 (CaCl2)

7.4 TEST METHOD

7.4.1    Control/Sample

7.4.1.1    Pipette (in milliliters) the following Reagents into suitable vials:

  Test Blank
Hem (Reagent 7.3.2) 2.50 2.50

 

7.4.1.2    Equilibrate to 37°C for 10 minutes. Then add:

  Test Blank
TCA (Reagent 7.3.4) ---- 5.00
Pro K (Reagent 7.3.9) 0.50 ----

 

7.4.1.3    Mix by swirling and incubate at 37°C for exactly 10 minutes. Then add:

  Test Blank
TCA (Reagent 7.3.4) 5.00 ----
Pro K (Reagent 7.3.9) ---- 0.50

 

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

7.4.1.5    7.4.1.5 Clarify by filtration through a 0.45 µm filter.

7.4.1.6    Add the following (in milliliters) in suitable containers:

  Test Blank
Filtrate/Supernatant (from step 7.4.1.5) 2.50 2.50
NaOH (Reagent 7.3.5) 5.00 5.00

 

7.4.1.7    Mix by swirling. Proceed with standard curve preparation.

7.4.2 Standard Curve

7.4.2.1    Prepare a standard curve by pipetting (in milliliters) the following reagents into suitable containers:

  Std 1 Std 2 Std 3 Std 4 Std 5 Blank
Std Soln (Reagent 7.3.7) 0.05 0.10 0.30 0.50 0.70 ----
HCl (Reagent 7.3.8) 2.45 2.40 2.20 2.00 1.80 2.50
NaOH (Reagent 7.3.5) 5.00 5.00 5.00 5.00 5.00 5.00

 

7.4.2.2    Mix thoroughly by swirling.

7.4.2.3    Add 1.50 mL of F-C (Reagent 7.3.6) to all standard, test, and blank tubes.

7.4.2.4    Immediately mix thoroughly by swirling or vortexing and incubate at room temperature for 30 min.

7.4.2.5    Transfer each test, blank, and standard solutions into suitable cuvettes and measure the absorbance at 750 nm of all solutions. If the solutions are hazy, filter through a 0.45 µm filter immediately prior to measuring.

7.5 CALCULATIONS

7.5.1    Standard Curve:

7.5.1.1    ΔA750nm Standard = A750nm Standard - A750nm Standard Blank

7.5.1.2    Plot the ΔA750nm Standard vs µmoles of Tyrosine.

7.5.2    Sample Determination:

7.5.2.1    ΔA750nm Sample = A750nm Test - A750nm Sample Blank

7.5.2.2    Determine the µmoles of Tyrosine equivalents liberated using the Standard curve.

7.5.2.3 Units/ml enzyme = (µmole Tyrosi ne equivalent s released) (8.0(df)
(0.5)(10)(2.5)

 

where:
    8.0 = Total volume (in milliliters) of stopped reaction
    10 = Time of assay (in minutes)
    0.50 = Volume of enzyme (in milliliter) of enzyme used
    2.5 = Volume (in milliliters) used in the Colorimetric Determination

7.5.2.4 Units/mg solid = Units/mL enzyme
mg solid/mL enzyme

 

7.5.2.5 Units/mg protein = Units/mL enzyme
mg protein/mL enzyme

 

7.6 FINAL ASSAY CONTENTRATION:
In a 3.00 mL reaction mix, the final concentrations are 83 mM potassium phosphate, 1.67% (w/v) Hemoglobin, 5.0 M urea, 3.33 mM calcium chloride, and 0.037 - 0.088 unit proteinase K.

8. References & Attachments

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

9. Approval

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