Inhibition of Acid Proteases by a Pepsin Inhibitor (S-PI)

Abstract

S-PI inhibited various acid proteases including pepsin, Rhodotorula glutinis acid protease and Cladosporium acid protease, but the rate of inhibition was different for each acid protease.
S-PI made an equimolar complex with these acid proteases. A part of the enzyme-S-PI complex dissociated in the reaction mixture and showed proteolytic activity. The specific activity of the enzyme-S-PI complex depended on the concentration of the complex in the reaction mixture. Compared with native (S-PI free) enzyme, each of the enzyme-S-PI complex showed 50% activity at the following concentrations, pepsin; 7.5×10^-10M, Rh. glutinis acid protease; 1.8×10^-7M, Cladosporium acid protease; 3.0×10^-6M.
These acid proteases were stabilized from heat or acid denaturation by making the enzyme-S-PI complex. S-PI protected the modification of these acid proteases by diazoacetyl-DL-norleucine methyl ester.
Binding between these acid proteases and S-PI dissociated at around neutral pH. S-PI was separated from enzyme-S-PI complex by dialysis at pH 7.5. In this case, pepsin underwent denaturation, while denaturations of Rh. glutinis acid protease and Cladosporium acid protease were slight. Rh. glutinis acid protease and Cladosporium acid protease were recovered from enzyme-S-PI complex by DEAE cellulose column chromatography as a native form.