Contributions of Histochemistry to the Development of the Proteolytic Enzyme Detection System in Diagnostic Medicine

Although the place of enzyme-catalyzed reactions in clinical biochemistry has proven to be of diagnostic value since the late 1950s (16,17), it is only within the last 6 years that methods for determining the activity of proteases have been introduced into the clinical laboratory. Proteolytic enzymes are assuming increasing significance in biological regulation. An understanding of their physiological and pathological roles has come from both biochemical and histochemical standpoints. Proteases, unlike most hydrolases, the general class of enzymes to which they belong, are highly specific and precisely controlled. Furthermore, when this precise control of proteolysis breaks down, the consequences are usually serious. It has long been recognized by physiological chemists that many disease states represent abnormalities of protein synthesis or degradation resulting from disturbances in the activity of proteases or their inhibitors. First attempts at assaying for specific proteases by preparing synthetic substrates were made by Fruton and Bergmann in the 1930s. They found that crude preparations from bovine spleen and partly purified preparations of pepsin and trypsin could hydrolyze small synthetic substrate molecules with free amines. The task for assaying protease activity was greatly simplified when in 1950, Neurath and Schwert demonstrated that amino acid esters were very sensitive substrates. Such substrates were used by Sherry and Troll (58) and associates in the mid-1950s to study clotting and fibrinolytic enzymes. It was Gomori, in 1954, who used chloroacetyl-/3-naphthylamine with ammonia to obtain glycine and D,L-alanyl-/3-naphthyl-