Proteinase K Assay

Method: Proteinase K hydrolyzes hemoglobin denatured with urea, and liberates Folin Postive amino acids and peptides, which are determined as tyrosine equivalents. 1 unit releases 1 μ mole of Folin positive amino acid at 37°C, pH 7.5, using denatured hemoglobin as substrate.

Reagents

  • 0.05 N HCl - Dilute 0.82 ml conc. HCl to 200 ml with reagent grade water.
  • 0.5 M NaOH - Dissolve 4.0 gm NaOH in 200 ml reagent grade water.
  • Buffer-Substrate - Dissolve 2.0 gm hemoglobin in 35 ml reagent grade water, add 36.0 gm urea and 16 ml 0.5 M NaOH. Stir for 30 - 60 minutes at room temperatue. Add 0.618 gm boric acid and stir. Adjust the pH to 7.5 with 5 N HCl and q.s. to 100 ml.
  • Tyrosine standard (2.5 nmol/L) - Dissolve 45.3 mg tyrosine in 100 ml of 0.05 N HCl.
  • 0.3 M Trichloroacetic acid - Dissolve 9.8 gm trichloroacetic acid in 200 ml reagent grade water.
  • Folin Reagent - Add 10 ml Folin-Ciocalteus Phenol Reagent to 20 ml reagent grade water.

    Enzyme

    Dissolve 10 mg lyophilized material in 1 ml reagent grade water. Prepare a 1:1000 dilution with water immediately before use.

    Procedure

    Label clear glass test tubes for blank, standard, and test. Add 2.5 ml buffer-substrate and incubate for 5 minutes at 37°C. Start reaction by adding 0.2 ml tyrosine standard to the standard tube, 0.2 ml of sample to the test, and 0.2 ml of 0.05 N HCl to the blank. Incubate for 10 minutes at 37°C. Stop reaction by the addition of 5.0 ml trichloroacetic acid. Mix, then add 0.2 ml of sample to the blank and standard, and add 0.2 ml of 0.05 N HCl to the test. Mix and let stand for 10 minutes at room temperature, filter and pipette into test tubes 1.0 ml of filtrate, 2.0 ml of 0.5 N NaOH, and 0.6 ml of Folin Reagent. Mix well. Let stand for 15 minutes and read A578 nm.

    Calculation

    calculation

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