Carboxypeptidase-B from Bubalus bubalis pancreas: Purification, properties and MALDI-TOF monitored activation of proinsulin

Carboxypeptidase-b (e.c 3.4.17.2) catalyzes the hydrolysis of peptides and esters at c-terminus of arginine and lysine residues. our study describes the large scale purification, n-terminal sequence analysis and physiochemical properties of pancreatic enzyme from river buffalo (bubalus bubalis). the enzyme was purified up to 71 folds by anionexchange chromatography with 21% final recovery. purified enzyme displayed two bands on sds-page with molecular weights of 9 kda and 26 kda respectively, the n-terminal sequence of later was efldkldfyv. the enzyme has shown optimum activity at ph 9.0 and 40◦c. the km, kcat and kcat/km values of purified carboxypeptidase-b with hippuryl-larg are 30μm, 72sec-1 and 2.4x105 m-1 sec-1 respectively. a computer based model for the structure of enzyme was proposed by chromatographic studies of component fragments and n-terminal sequence. the enzyme purified in the present study was free of carboxypeptidase a and endoprotease contamination. it was efficiently used in the processing of recombinant buffalo proinsulin, in combination with trypsin. activation of proinsulin was monitored by maldi-tof analysis of peptides before and after the action of enzymes.