PEPTIDYL METHYL KETONES AS LIGANDS IN AFFINITY-CHROMATOGRAPHY OF SERINE AND CYSTEINE PROTEINASES

Peptidyl methyl ketones are reversible inhibitors of serine and cystei ne proteinases and can be employed as ligands in affinity chromatograp hy. Serine proteinases of the subtilisin family may be purified using the tripeptidyl methyl ketone -Ala2-PheCH3 as ligand, whereas for papa in, the best-known cysteine proteinase, both the -Ala2-PheCH3 and the Phe-AlaCH3 ligands are efficient. Complete elution of affinity-bound p roteinases with isopropanol was demonstrated by investigations with th ermitase, a subtilisin-type serine proteinase, which was C-14 labelled . Bound proteinases with different affinity for a defined ligand may b e consecutively eluted using increasing concentrations of isopropanol in the elution buffer, as was shown, for example, with a mixture of th ermitase and subtilisin DY. The quality of the lyophilized affinity-pu rified thermitase is similar to that of thermitase preparations purifi ed by isoelectric focusing and adsorption on porous glass bodies. The affinity of thermitase for the immobilized -Ala2-PheCH3 ligand linked to Divicell via an epsilon-aminocaproic acid (Aca) spacer is about two orders of magnitude lower than its affinity for the soluble inhibitor benzyloxycarbonyl-Ala2-PheCH3 and one order of magnitude lower than i ts affinity for Ac-Aca-Ala2-PheCH3. In comparison with affinity gels m ade with commercial CH-Sepharose 4B, the Divicell gels possess higher concentrations of ligands and have a higher stability.