Covalent Chromatography for Chymotrypsin-like Proteases Using a Diphenyl 1-Amino-2-phenylethylphosphonate Derivative

To establish a covalent chromatography system for purification of naturally occurring chymotrypsin-like serine proteases, a diphenyl 1-amino-2-phenylethylphosphonate derivative bearing Gly-Gly-Gly as a spacer was immobilized on the Sepharose 4FF gel. In this system, bovine chymotrypsin was selectively bound to the phosphonate immobilized on the gel and then released by the action of 2-pyridinealdoxime methiodide as the reactivated enzyme. Based on the study results for selective binding and reactivation conditions, chymotrypsin-like proteases from pancreatin (hog pancreas) were rapidly and highly purified within three hours. Keywords