Determination of proteolytic activity using l-[4,5-3h]leucine-labelled globin as a substrate

Abstract

A method is described for the assay of proteolytic activity, based on the digestion of L-[4,5-3H]leucine globin. L-[4,5-3H]Leucine was incorporated into the substrate at the stage of haemoglobin biosynthesis, using rabbit erythrocytes. Assay methods for proteolytic enzymes have been based on the digestion of haemoglobin, serum albumin or casein, and the determination of the trichloroacetic acid-soluble products [1,2]. More sensitive methods have been developed by using haemoglobin labelled with a fluorescent [3-5] or radioactive marker [6,7]. These methods avoid the errors which beset the Anson procedure, such as interference by impurities (purines at 280 nm and reducing compounds at 700 nm) [8]. However, methods using labelled proteins as a substrate present a number of problems, the most troublesome of which are the high blank values and the use of non-physiological substrates when chemically modified proteins are employed. In the present communication a simple and sensitive method for the assay of proteolytic enzyme activity is described. This is based on the digestion of L-[4,5-3H]leucine globin by proteolytic enzymes and radioactivity measurement of the trichloroacetic acid soluble cleavage products.