Chromogenic Substrates

 

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CHROMOGENIC SUBSTRATES | METHODS | CHYMOTRYPSIN METHOD


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 (DA/min) or absorbance (A) and the chymotrypsin activity is linear in the 0.05-1.0 kat/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 Chymotrypsin
MeO-Suc-Arg-Pro-Tyr-OH + pNA

Reagents

  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 (25C)
  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 25C 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 2-8C.

  1. Acetic acid 20%
    Acetic acid is used in the acid-stopped method.

Sample
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 2-8C. 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.

Method

Initial rate method
Buffer 200 ml
Incubate at 37C 3-4 min
Chymotrypsin sample 200 ml
Mix and incubate at 37C 2-3 min
Substrate 200 ml

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

method

Sample

Blank

Buffer

200 ml

200 ml

Incubate at 37C

3-4 min

-

Chymotrypsin sample

200 ml

200 ml

Mix and incubate at 37C

2-3 min

-

Substrate (37C)

200 ml

-

Mix and incubate at 37C

3 min

-

Acetic acid 20%

200 ml

200 ml

Mix

yes

-

Substrate

-

200 ml

Mix 

-

yes

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

Calculation

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

Bibliography

  1. Tzsr 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)
 

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