Reversed-phase high-performance liquid chromatography of peptides of porcine pepsin prepared by the use of various forms of immobilized α-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 α-chymotrypsin. Porcine pepsin was hydrolysed with soluble α-chymotrypsin, with α-chymotrypsins glycosylated with lactose or galactose coupled to hydrazide derivative of cellulose, with α-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 α-chymotrypsins immobilized by different methods. Best results were achieved after hydrolysis with α-chymotrypsin immobilized on poly(acrylamide-allyl glycoside) copolymers. α-Chymotrypsin immobilized by this way has further three times higher relative specific activity in comparison with the soluble one. Modified α-chymotrypsin was not suitable for efficient pepsin cleavage.