An immobilized naphthylamide substrate for proteinases with tryptic-like specifleity

Abstract

An immobilized amino acid naphthylamide substrate for proteinases with tryptic-like specificity was prepared by reacting -arginine [beta]-naphthylamide with an N-hydroxysuccinimide-activated derivative of agarose. Hydrolysis of the immobilized substrate (A10-Arg-[beta]NA) was followed by monitoring the increase in fluorescence accompanying the release of [beta]-naphthylamine. Assays using A10-Arg-[beta]NA were designed for quantitatively determining the presence of 1-2 pmol of trypsin and 15 pmol of thrombin. Profibrinolysin, fibrinolysin, and urokinase have either no or very low activities with A10-Arg-[beta]NA. Trypsin complexed with [alpha]2-macroglobulin has no activity (2-macroglobulin titrations was demonstrated.