Abstract
Acid stable trypsin inhibitor (ASTI) enzymatically produced from human plasma precursor protein (pro-inhibitor) with bromelain was highly purified by trypsin-Sepharose affinity chromatography, isoelectric focusing and gel filtration on Sephadex G-75 superfine. The purified inhibitor was thought to be a single chain molecule and had an isoelectric point (pI) of 3.7, a molecular weight of 16, 000 by gel filtration (specific activity 3, 600U/mg protein), and was a Gly- and Glu-rich protein lacking His. The NH2-terminal amino acid sequence determined by automated gas-phase sequence analyzer was H2N-(Lys)-Glu-Asp-Ser-Cys-Gln-Leu-Gly-Tyr-Ser-Ala-Gly-Pro-X-Met-Gly-Met-…COON, which was homologous to the Lys21-Met31 part (or Lys22-Met31 part: 30% of the total) of the urinary ASTI (UTI) molecule (p1=2.0; mol. wt. 67, 000 by gel filtration). Both purified inhibitors exerted strong inhibitory effects on plasmin amidolysis (H-D-Val-Leu-Lys pNA; Ki=1.13-2.42×10-7M), but much lesser effects on plasmin fibrinolysis using standard fibrin plate method. They also strongly inhibited non-plasmic fibrinolysis with human leukocyte protease.
