Immobilization of α-chymotrypsin to magnetic particles and their use for proteolytic cleavage of porcine pepsin A

To detect phosphorylation state of pepsin, a simple and a rapid procedure for coupling of α-chymotrypsin to commercially available magnetic particles was elaborated. α-Chymotrypsin was immobilized to –CHO activated commercial magnetic particles via the protein free amino groups. The following properties of the immobilized proteinase were compared with those of the soluble enzyme: pH dependence of the activity, thermo-stability, self-cleavage activity, and possibility of repeated use.The immobilized α-chymotrypsin was used to study the phosphorylation degree of porcine pepsin A, used as a model acidic protein and phosphoprotein. The use of enzyme immobilized to magnetic carriers has several advantages as compared with an application of soluble forms of enzymes: preferably an increased stability of enzymes, a possibility of direct use of enzyme reaction products for MALDI-TOF MS. The prepared proteolytic digest was separated using RP-HPLC and immobilized metal affinity chromatography (IMAC) with immobilized Fe(III) ions prior to MALDI-TOF analysis: the presence of phosphopeptide in porcine pepsin A was shown.