Muhammad Shahid Nadeem, Bibi Nazia Murtaza, Habib Ahmad.
Carboxypeptidase-b from Bubalus bubalis pancreas: purification, properties and MALDI-TOF monitored activation of proinsulin.
Pak J Pharm Sci Jan ;26(5):907-13.
Carboxypeptidase-B (E.C 220.127.116.11) 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 anion-exchange 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-L-Arg 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.
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