An immobilized naphthylamide substrate for proteinases with tryptic-like specificity.

An immobilized amino acid napbthylamide substrate for proteinases with tryptic-like specificity was prepared by reacting L-arginine P-naphthylamide with an N-hydroxysuccinimide-activated derivative of agarose. Hydrolysis of the immobilized substrate (AlO-Arg-PNA) was followed by monitoring the increase in fluorescence accompanying the release of /3-naphthylamine. Assays using AIO-Arg-PNA were designed for quantitatively determining the presence of l-2 pmol of trypsin and 15 pmol of thrombin. Profibrinolysin, fibrinolysin, and urokinase have either no or very low activities with AlO-Arg-PNA. Trypsin complexed with a,-macroglobulin has no activity (~0.5%) toward AlO-Arg-PNA. The use of AlO-Arg-PNA for monitoring trypsin-a,-macroglobulin titrations was demonstrated.