Polyacrylamide Gel Entrapment of Adenylate Kinase and Acetate Kinase

The factors that limit the stability of adenylate kinase and acetate kinase in solution have been examined and compared with those that determine stability under conditions encountered during photochemically initiated polymer gel formation in solutions of acrylamide and N,N'-methylene-bisacrylamide. Both adenylate kinase (from rabbit and pig muscle) and acetate kinase (from E. Coli) contain cysteine residues close to their active sites. In solutions exposed to air, the rate of deactivation of these enzymes is determined by the rate of autoxidation (probably transition metal-catalyzed) of thbir cysteine sulfhydryl groups. Both enzymes are very stable if protected against autoxidation. At least four types of reactions contribute to deacti-vation during polyacrylamide gel formation: autoxidation of cysteine sulf-hydryl groups by molecular oxygen; Michael addition of cysteine thiolate anion to acrylamide monomer and related electrophilic species;reaction of cysteine and of other amino acids with singlet oxygen (generated by energy transfer from excited riboflavin to ground-state molecular oxygen during irradiation);reaction of several amino acid residues with free radicals (presumably SOl or buffer-derived radicals). To avoid deactivation during acrylamide polymerization, it is helpful to exclude molecular oxygen, to work at low temperature and low pH, and to add thiols to the solution as radical scavengers. Both enzymes are less susceptible to deactivation in solutions having high concentrations of substrates. Aciditional protection against singlet oxygen is afforded by using a tertiary amine buffer, and by adding pcarotene to the solution; both are effective quenchers for singlet oxygen. Adenylate kinase and acetate kinase have been modified by converting their cysteine-SH groups to-SSCH3 moieties by reaction with S-methyl methanethiosulfonate; this blocking is completely reversed by treatment with DTT. These modified proteins show 70% and \OVo, respectively, of the activity of the native enzymes. They are much more resistant to autoxida-tion and Michael addition than are the native proteins;their resistance to 178 singlet oxygen is slightly better than these proteins; their resistance to de-activation by SO; radical is indistinguishable from that of the fully reduced precursors. By taking advantage of a detailed accounting of the course of deactivation during polyacrylamide gel formation, it is possible to design experimental procedures that allow cross-linked polyacrylamide gels to be formed by free-radical polymerization in solutions containing adenylate kinase with preservation of 50-90% of the activity of the enzyme, and in solutions containing acetate kinase with preservation of 25-60% of the activity of the enzyme. If protected from atmospheric oxygen, the enzymes remain active in …