chromozym-pl and 4-nitroaniline

Guerin epithelioma, a highly metastatic tumour implanted to Wistar rats contains two inhibitors of fibrinolysis which can be detected with the use of zymographic techniques. The first one--with Mr about 48000 forms SDS-stable complex with urokinase. The second--with Mr about 7000 inhibits fibrinolytic and amidolytic activity of plasmin.

Topics: Aniline Compounds; Animals; Antifibrinolytic Agents; Carcinoma; Chromogenic Compounds; Electrophoresis, Polyacrylamide Gel; Female; Fibrin; Fibrinolysin; Molecular Weight; Neoplasm Transplantation; Neoplasms, Experimental; Oligopeptides; Rats; Rats, Inbred Strains; Urokinase-Type Plasminogen Activator

We describe an enzymic assay for the functional activity of alpha 1-antitrypsin, in which tosyl-Gly-Pro-Lys-p-nitroanilide acetate (Chromozym PL) is the trypsin substrate. For the indicating reaction, the concentration of substrate is about double the Km of its reaction with trypsin. This assay is an improvement over reactions involving N-alpha-benzoyl-DL-arginine p-nitroanilide as substrate, the solubility of which in water is less than the Km of its reaction with trypsin. The assay is standardized in terms of active sites of trypsin inhibited per liter of serum, with p-nitrophenyl-p'-guanidinobenzoate as the active site titrant. The results agreed well with those by an immunonephelometric assay for alpha 1-antitrypsin (r = 0.953). The within-run and run-to-run precision was approximately 3%. The results of the assay varied linearly with alpha 1-antitrypsin concentration from 0 to 60 mumol/L of serum. The minimum sample size was 50 microL, and analysis of 24 samples took less than 20 min.

Topics: alpha 1-Antitrypsin; Aniline Compounds; Autoanalysis; Centrifugation; Chromogenic Compounds; Humans; Kinetics; Nephelometry and Turbidimetry; Oligopeptides; Spectrophotometry; Trypsin