On the Characterization of Porcine Erythrocyte Catalase and Its Disulfide-Linked Dimer

抄録

Purified native erythrocyte catalase [EC 1. 11. 1. 6] (monomer) associates into dimer, trimer, tetramer and larger molecules by forming disulfide cross-links due to air oxidation of SH groups in the protein moiety. The monomer and dimer could be isolated independently from the other components by gel filtration on a Bio-gel column. The sedimentation constants (s⁰_{20, w}) of the monomer and dimer were 11.6 and 15.9 S, respectively, which correspond to molecular weights of 280, 000±14, 000 and 550, 000±27, 500 as estimated by the sedimentation equilibrium method. From these values, the frictional ratios of monomer and dimer were calculated to be 1.33 and 1.51, respectively. The specific activity of the enzyme was almost unchanged upon dimerization. The dimer contains 8 heme groups per molecule in contrast to 4 heme groups in a monomer molecule, confirming that dimerization had occurred. Amino acid analysis and spectrophotometric titration with p-chloromercuribenzoate revealed that only one free SH group out of 14 half-cystines in the monomer was directly available to form a disulfide cross-link upon dimerization. CD spectra of both monomer and dimer showed a clear positive CD band at 420nm as well as a prominent negative band in the Soret absorption region, quite different from the spectrum observed for bovine liver catalase. There was no marked difference between the monomer and dimer in absorption and CD spectra. The MCD spectrum of the dimer was significantly different from that of the monomer. The effects of pH on the activity and stability of both monomer and dimer were studied in terms of α-helical conformation as well as fluorescence of tryptophanyl residues and 8-anilinonaphthalene-1-sulfonate (ANS) bound to catalase molecules. From these results, together with results on the stability of the enzyme activity to heat and denaturants, we conclude that the dimer is more stable than the monomer due to the disulfide cross-link formed between monomer molecules upon dimerization.