Characterization of the dimer-monomer equilibrium of the papaya Copper/Zinc superoxide dismutase and its equilibrium shift by a single amino acid mutation.

摘要: The coding region of the copper/zinc superoxide dismutase (Cu/Zn SOD) cDNA from papaya fruit, Carica L. cv. Tainong 2, was cloned into an expression vector, pET-20b(+). Cu/Zn SOD expressed in Escherichia coli and purified by His-tag technique. Two active forms enzyme (30% dimer 70% monomer) equilibrium were observed. activity dimeric higher than that monomeric form. thermal inactivation rate constant K(d) values calculated for monomer at 90 degrees C -0.0203 -0.0216 min(-1), half-lives 41.9 31.8 min, respectively. This indicated more stable its dimerization inhibited under acidic pH (below 3.0) or imidazole buffer (above 0.5 M), whereas it not affected alkaline 9.0). Both 1-4% SDS. Furthermore, much resistant to proteolytic attack after 3 h incubation 37 with trypsin chymotrypsin. In addition, mutation position 48 Leu Phe (L48F) association monomer, a mutant Lys substitution (L48K) same tended dissociate