Determination of chymotrypsin activity with Chromogenic Substrate S-2586

Measurement Principle
The chymotrypsin activity is determined by its amidolytic effect on the substrate MeO-Suc-Arg-Pro-Tyr-pNA (S-2586). The rate at which p-nitroaniline (pNA) is released is measured photometrically at 405 nm. This can be followed on a recorder (initial rate method) or read after stopping the reaction with acetic acid (acid stopped method). The correlation between the change in absorbance per minute (ΔA/min) or absorbance (A) and the chymotrypsin activity is linear in the 0.05-1.0 µkat/l or 3-60 U/l range. The amidolytic activity of different chymotrypsin preparations does not necessarily parallel the protease activity.

MeO-Suc-Arg-Pro-Tyr-pNA + H2O   →   MeO-Suc-Arg-Pro-Tyr-OH + pNA


  1. Chromogenic Substrate S-2586, 25 mg Art. No. 82 08 94
    Reconstitute the substrate S-2586 (MW: 705.3) with 60 ml of distilled water.
  2. Buffer – Tris/Calcium, pH 8.3 (25°C)
  3. Tris 12.1 g (100 mmol/l)
    NaCl 56.2 g (960 mmol/l)
    Distilled water 800 ml
    Adjust the pH to 8.3 at 25°C by adding approximately 50 ml of 1 mol/l HCl. Add 10 ml of 1 mol/l CaCl 2 solution. Fill up to 1000 ml with distilled water. The buffer, if not contaminated, will remain stable for two months at 2-8°C.
  4. Acetic acid 20%
    Acetic acid is used in the acid-stopped method.


The sample containing chymotrypsin is dissolved in or diluted with 1 mmol/l HCl to a concentration of 0.1 g/l. This stock solution is stable for more than two weeks at 2-8°C. Before assay, the solution is diluted 1:200 with 1 mmol/l HCl. If the sample is a pure protein, it is advisable to use 0.1% Carbowax 6000 (Union Carbide, NY) or 1% albumin (previously checked for amidolytic activity) to avoid adsorption to surfaces.


Initial rate method

Header 1 Header 2
Buffer 200 µl
Incubate at 37°C 3-4 min
Chymotrypsin sample 200 µl
Mix and incubate at 37°C 2-3 min
Substrate 200 µl

Transfer immediately to a 1 cm semi-microcuvette (preheated to 37°C) for measurement of the absorbance change in a photometer at 405 nm and at 37°C, calculate ΔA/min.

method Sample Blank
Buffer 200 ml 200 µl
Incubate at 37°C 3-4 min
Chymotrypsin sample 200 ml 200 µl
Mix and incubate at 37°C 2-3 min
Substrate (37°C) 200 ml
Mix and incubate at 37°C 3 min
Acetic acid 20% 200 ml 200 µl
Mix yes
Substrate 200 µl
Mix yes

Read the absorbance (A) of the sample against distilled water at 405 nm within 4 hours.


Calculate the chymotrypsin activity of the stock solution from the following formulas:
Initial rate method:
µkat/l = 5.19 x ΔA/min x 200
U/l = 311 x ΔA/min x 200
Acid stopped method:
µkat/l = 2.31 x A x 200
U/l = 138 x A x 200


  1. Tözsér J et al. Active centre studies on bovine pancreatic chymotrypsin with tripeptidyl-p-nitroanilide substrates. Acta Biochim Biophys Hung 21, 335-348 (1986).
  2. Takayama TK et al. Characterization of the precursor of prostate-specific antigen. Activation by trypsin and by human glandular kallikrein. J Biol Chem 272, 21582-8 (1997).
  3. Pejler G, Sadler JE. Mechanism by which heparin proteoglycan modulates mast cell chyma activity. Biochemistry 38, 1287-95 (1999)