Reversed-phase high-performance liquid chromatography of peptides of porcine pepsin prepared by the use of various forms of immobilized alpha-chymotrypsin.
Reversed-phase high-performance liquid chromatography (RP-HPLC) separation was used for the comparison of peptide maps of pepsin after its digestions by different forms of immobilized alpha-chymotrypsin. Porcine pepsin was hydrolysed with soluble alpha-chymotrypsin, with alpha-chymotrypsins glycosylated with lactose or galactose coupled to hydrazide derivative of cellulose, with alpha-chymotrypsin attached to poly(acrylamide-allyl glycoside) copolymer or to glycosylated hydroxyalkyl methacrylate copolymer Separon or to agarose gel Sepharose 4B. Efficiency of enzymatic protein cleavage with regard to peptide mapping of porcine pepsin has been examined by the use of alpha-chymotrypsins immobilized by different methods. Best results were achieved after hydrolysis with alpha-chymotrypsin immobilized on poly(acrylamide-allyl glycoside) copolymers. Alpha-chymotrypsin immobilized by this way has further three times higher relative specific activity in comparison with the soluble one. Modified alpha-chymotrypsin was not suitable for efficient pepsin cleavage.
Vanková
H
H
Institute of Pathophysiology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic. hvank@lfl.cuni.cz
Kucerová
Z
Z
Turková
J
J
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002918:Chymotrypsin / Q000737:chemistry*; D010434:Pepsin A / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification*; D010449:Peptide Mapping; D013056:Spectrophotometry, Ultraviolet; D013552:Swine
D010446:Peptide Fragments; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D010434:Pepsin A