Purification and characterization of a sulfhydryl-dependent protease from Rhodnius prolixus midgut

Abstract

The characterization and partial purification of one protease from Rhodnius prolixus midgut are described. Enzyme preparations show a high degree of instability under several conditions, such as handling, freezing, and thawing, and at 4°C. This spontaneous inactivation of either crude or purified enzyme is completely protected by 10 mM tetrathionate. Sulfhydryl compounds such as β-mercaptoethanol, dithiothreitol, cysteine, thioglycolic acid, and reduced glutathione are able to activate the enzyme, whereas iodoacetate, mercuric chloride, p -aminophenylmercuric chloride, tetrathionate, and 5,5-dithiobis(2-nitrobenzoic acid) inhibit the enzyme. Partial inhibition was obtained by hydrogen perioxide, oxygen bubbling, and performic acid. Besides casein, the enzyme hydrolyzed benzoyl- l -tyrosine ethyl ester (BTEE), but showed no esterolytic activity upon p -tosyl- l -arginine methyl ester and benzoyl- l -arginine ethyl ester. Purifications of 210- and 187-fold with yields of 80 and 71% respectively, were obtained for the caseinolytic and esterolytic activities by using a Sepharose-mercurial column as a thiol-group affinity adsorbent. The molecular weight was estimated as 21,500 by gel filtration. α-Chymotrypsin and the midgut enzyme show similar abilities to hydrolyze casein, however, the midgut protease hydrolyzes casein 2.7-fold faster than trypsin and about 3-fold slower than pepsin. Soybean trypsin inhibitor, black-eyed pea chymotrypsin inhibitor, tosyl- l -lysine chloromethyl ketone, and tosyl-amide-phenylethyl chloromethyl ketone do not inhibit the protease activity, whereas, pepstatin showed a 100% inhibitory effect at an inhibitor/enzyme molar ratio of 0.36. Midgut protease and α-chymotrypsin showed similar K m values for BTEE (1.06 and 1.15 m ×10 −4 , respectively), however, the former showed a 9-fold lower catalytic efficiency ( K cat / K m ). Dialysis did not affect the enzyme, whereas Cu 2+ , Pb 2+ , and Hg 2+ showed an inhibitory effect; a slight activation was obtained in the presence of Ca 2+ .