Transition-state affinity chromatography of trypsin-like proteinases with dipeptidyl argininal ligands.
Dipeptidyl argininal (arginine aldehyde) affinity resins of general formula R-(X-Y-argininal) (where R = resin matrix and X, Y = amino acids of varied structure) are synthesized in a solid-phase procedure in which the dipeptide (-X-Y-) is first attached to the resin, followed by the joining of the Y amino acid to argininal semicarbazone, and decomposition of the semicarbazone in a methanol/acetic acid/formaldehyde reagent. An R-(Gly-Gly-argininal) resin binds urokinase tightly, but does not bind thrombin. However, thrombin binds strongly to R-(Phe-Pro-argininal), whereas urokinase does not bind. Accordingly, the X-Y-argininal ligands selectively bind proteinases of identical primary binding site specificity to arginine, but different secondary site specificity in -X-Y-. The selectivity is due to an amplification of peptide binding specificity caused by the transition-state analog properties of the ligands. While the affinity constants between peptide aldehyde and proteinase approach those of antibody-antigen interactions, the elution with semicarbazide (aldehyde-trapping reagent) buffers easily remove tightly bound proteinases without proteinase inhibitors or denaturation. Conditions for the binding and elution of proteinases, methods of regeneration and other characteristics of the resins are described.
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D002918:Chymotrypsin / Q000302:isolation & purification*; D004151:Dipeptides; D006801:Humans; D008024:Ligands; D013917:Thrombin / Q000302:isolation & purification*; D014357:Trypsin; D014568:Urokinase-Type Plasminogen Activator / Q000302:isolation & purification*
D004151:Dipeptides; D008024:Ligands; C033879:argininal; D001120:Arginine; D002918:Chymotrypsin; D014357:Trypsin; D013917:Thrombin; D014568:Urokinase-Type Plasminogen Activator