title: Carboxypeptidase-B from Bubalus bubalis pancreas: purification, properties and MALDI-TOF monitored activation of proinsulin.
abstract: 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 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 2.4x10(5) 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.
authors.affiliation: Department of Genetics Hazara University Garden Campus Mansehra 21300, Pakistan.
authors.forename: Muhammad Shahid
authors.initials: MS
authors.lastname: Nadeem
authors.forename: Bibi Nazia
authors.initials: BN
authors.lastname: Murtaza
authors.forename: Habib
authors.initials: H
authors.lastname: Ahmad
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002020:Buffaloes; D043424:Carboxypeptidase B / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D003198:Computer Simulation; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology*; D011384:Proinsulin / Q000378:metabolism*; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
chemical_list: D007328:Insulin; D011384:Proinsulin; D043424:Carboxypeptidase B; D014357:Trypsin