Carboxypeptidase-beta from Bubalus bubalus pancreas: purification, properties and MALDI-TOF monitored activation of proinsulin
Pakistan Journal of Pharmaceutical Sciences. 2013; 26 (5): 907-913
in English
| IMEMR
| ID: emr-138408
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 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 K[M], Kcat and K[cat]/K[M] values of purified carboxypeptidase-B with Hippuryl-LArg are 30 micro M, 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
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Index:
IMEMR (Eastern Mediterranean)
Main subject:
Pancreas
/
Proinsulin
/
Structure-Activity Relationship
/
Substrate Specificity
/
Temperature
/
Computer Simulation
/
Buffaloes
/
Molecular Sequence Data
/
Kinetics
/
Models, Molecular
Limits:
Animals
Language:
English
Journal:
Pak. J. Pharm. Sci.
Year:
2013
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