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 h ydrolysed with soluble alpha -chymotrypsin, with alpha -chymotrypsins glyco sylated with lactose or galactose coupled to hydrazide derivative of cellul ose, 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 o f alpha -chymotrypsins immobilized by different methods. Best results were achieved after hydrolysis with alpha -chymotrypsin immobilized on poly(acry lamide-allyl glycoside) copolymers. alpha -Chymotrypsin immobilized by this way has further three times higher relative specific activity in compariso n with the soluble one. Modified alpha -chymotrypsin was not suitable for e fficient pepsin cleavage. (C) 2001 Elsevier Science B.V. AU rights reserved .