DiaPharma Group, Inc.

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For research use only

S-2586 is a chromogenic substrate for chymotrypsin. The substrate has hitherto been used for the assay of purified preparations and for the assay of antichymotrypsin in blood plasma. It has also been used for the assay of chymotrypsin-like activity in other biological samples. It may also be used for the assay of chymotrypsin in pancreatic juice.

Reagent -- Composition, Packaging, Storage and Stability

Each vial contains chromogenic substrate S-2586 25 mg and mannitol 40 mg as a bulking agent.

Chemical name: 3-Carbomethoxypropionyl-Larginyl-L-prolyl-L-tyrosinep-nitroaniline hydrochloride

S-2586 structure

Formula: MeO-Suc-Arg-Pro-Tyr-pNA·HCl

Mol. wt.: 705.3

e316nm: 1.27·104 mol-1·L·cm-1

Solubility: > 10 mmol/l in H2O

Stability: Substance: Stable until expiry date if stored at 2-8°C. Avoid exposure to light. The substance is hygroscopic and should be stored dry.

Solution: 1 mmol/l in H2O is stable for more than one month at 2 to 8°C. Contamination by microorganisms may cause hydrolysis. Suitable stock solution: 1 mmol/l in H2O

Princpiple

	Enzyme
MeO-Suc-Arg-Pro-Tyr-pNA		MeO-Suc-Arg-Pro-Tyr-OH+pNA

The method for the determination of activity is based on the difference in absorbance (optical density) between the pNA formed and the original substrate. The rate of pNA formation,i.e. the increase in absorbance per second at 405 nm, is proportional to the enzymatic activity and is conveniently determined with a photometer.

Kinetic Data

Chymotrypsin: Km= 5·10-5 mol/l and Kcat= 140 sec-1. (Mol.wt. 25000. The enzyme is assumed to be pure.) Determined in 0.03 mol/l Tris pH 8.3, I 0.4 with 3 mmol/l CaCl2 and at 37°C.

Standardization

An activity of DA/min = 0.05 (37°C) is obtained by using a substrate concentration of 2·Km and 0.03 mg/l of alpha-Chymotrypsin Worthington (65 micro BTEE/mg) and similar activities were obtained by using enzymes from other sources.

Selectivity

The substrate is not split by trypsin, thrombin, factor Xa, plasma or glandular kallikreins, plasmin or urokinase at concentrations that readily split (DA/min =1) commercial-Arg-pNA substrates. Granulocyte elastase has very low activity on the substrate. Cathepsin G is known to split this type of substrates, but the activity per mg of enzyme is in the order of one per cent of that of chymotrypsin. Pancreas elastase free from chymotrypsin impurities does not split the substrate.