Principles of enzyme stabilization V. The possibility of enzyme selfstabilization under the action of potentially reversible intramolecular cross-linkages of different length

Abstract

The denaturing action of guanidine · HCl on modified α-chymotrypsin (EC 3.4.21.1) preparations has been studied. The consecutive treatment of α-chymotrypsin with N- acetyl-homocysteine thiolactone, 5,5′-dithio-bis-(2-nitrobenzoic acid) and dithiols of HS-( CH 2) n -SH type, with n ranging from 4 to 10, leads to enzyme stabilization as a result of protein modification. A greater stabilization effect can be achieved by enriching the protein molecule with groups reactive towards dithiols, after first modifying carboxygroups. In this case dithiol with n = 5 forms an intramolecular cross-linkage. If an equimolecular mixture of different dithiols is used for enzyme modification, the enzyme gradually ‘selects’ 1,5-dithiol for the formation of an intramolecular cross-linkage instead of the initial one-point modification. The use of potentially reversible cross-linkages may be generally employed for the preparation of stabilized water-soluble enzymes via the mechanism of selfstabilization.