Solid-phase active site inhibitors of proteinases.
Phenylalanine chloromethyl ketone covalently attached to porous glass beads was synthesized to serve as a solid-phase active site directed inhibitor of chymotrypsin-like proteolytic enzymes. The solid-phase reagent inhibited 20 nmol of bovine chymotrypsin per gram of glass and covalently bound 30 nmol of protein per gram of glass. Sepharose-bound lysine chloromethyl ketones were synthesized to serve as inhibitors of trypsin-like enzymes. Sepharose-MethionylLysyl chloromethyl ketone inactivated and bound about 6.8 nmol of enzyme per ml of settled gel. In a preliminary experiment, a cyanogen bromide cleavage of the methionine residues showed that it should be possible to release all peptides but the peptide containing the active-site histidine. The immobilized trypsin was also reduced, carboxymethylated and digested with chymotrypsin. The potential of the solid-phase approach is in the isolation of a specific serine proteinase and in the sequence determination of residues surrounding the active-site histidine.
D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D003488:Cyanogen Bromide / Q000494:pharmacology; D007659:Ketones; D011480:Protease Inhibitors / Q000494:pharmacology*
D007659:Ketones; D011480:Protease Inhibitors; D002918:Chymotrypsin; D003488:Cyanogen Bromide