Improved Assay Method of the Proteases in an Acidic Condition Using a-Naphthylester Substrates

Abstract

Recenity, we, developed the new synthetic substrates for various proteases, α-naphthylester derivatives. Principle of the assay method with these substrates was based upon diazo-coupling reaction between α-naphthol released by the enzyme reaction and diazonium salt. Among the various diazonium salts, Fast Violet B salt has been used in this assay system. However, Fast Violet B salt was possible to use only in neutral pH region, and in the absence of reducing or oxidizing reagent.
In this work, to establish the methods of microassay and zymogram on the proteases which have an optimum in acidic pH region, we tested the reactivities between a-naphthol and two diazonium salts, Fast Red ITR salt and Fast Blue BB salt. Consequently, only Fast Red ITR salt was found to react in acidic pH region (pH 4.0). λmax and ε values of the diazo-color with Fast Red ITR salt in acidic pH region were 475nm and 26, 000, respectively. Further, although the reaction between Fast Red ITR salt and α-naphthol was inhibited by the reducing reagent such as dithiothreitol, its inhibition was found to be removed in the coexistence of oxidizing reagent. These results indicate that, if Fast Red ITR salt is used as coloring reagent, α-naphthylester substrate is very effective for the investigation of lysosomal protease such as cathepsin B like enzymes.
Zymogram on lysosomal protease of rat liver was prepared in acidic pH region using Pro-Phe-Arg-α-naphthylester as the substrate and Fast Red ITR salt. Two bands were visualized, and the appearance of these bands was promoted in the presence of dithiothreitol, and completely inhibited by the addition of leupeptin. Therefore, these bands may be due to cathepsin B like enzyme.