A simple and sensitive proteinase assay using sepharose 4b-coupled fluorescamine-labeled casein as a substrate

A simple and sensitive method for proteinase assay was developed, which uses fluorescamine-labeled casein-Sepharose 4B as a substrate. Casein-Sepharose 4B was prepared most effectively by coupling casein to cyanogen bromide-activated Sepharose 4B at pH 10.0. Fluorescamine-labeled casein-Sepharose 4B was then prepared by mixing fluorescamine and casein-Sepharose 4B suspension at pH 8.0 and used for the assay as a substrate after removal of the excess reagent and/or its hydrolysis products. The assay can be done by simply measuring the fluorescence (excitation at 390 nm and emission at 475 nm) of the filtrate of the assay mixture after incubation of the substrate with enzyme solution.This method is suited for the assay of proteinases active at neutral to slightly alkaline pH values, and the activity of 3 ng of trypsin or 10 ng of α-chymotrypsin can be determined with reasonable accuracy. This method is therefore almost as sensitive as those using radioisotope-labeled proteins as substrates.