Dissociation of dimer of bovine erythrocyte Cu,Zn-superoxide dismutase and activity of the monomer subunit: effects of urea, temperature, and enzyme concentration.
Cu,Zn-superoxide dismutase (SOD) of bovine erythrocytes is a dimeric enzyme of identical subunits associated through unusually strong noncovalent interactions. It is known not to be dissociated into subunits even in 8 M urea for 72 h at 25 degrees C [Malinowski, D.P. & Fridovich, I. (1979) Biochemistry 18, 5055-5060]. Effects of urea, temperature, and SOD concentration on the inactivation and dissociation into subunits were examined. The activation energy of the inactivation of SOD (at 0.05 mg/ml) was 64 kcal/mol at pH 7.8, and was decreased to 40 kcal/mol in the presence of urea (2.0-7.3 M). The apparent first-order rate constant (kapp) of the inactivation by urea was dependent on the SOD concentration [SOD] during the urea treatment, and SOD showed a higher resistance to the inactivation with increase in the concentration. Dissociation of SOD was monitored by gel filtration HPLC. When SOD solutions of various concentrations were incubated in 6 M urea at 45 degrees C, two monomer species (M1 and M2) were observed in addition to dimer (D). The dimer maintained full activity, while the monomers did not show the activity. The peak areas of these species were changed depending on the SOD concentration during urea treatment; at over 15 mg/ml, almost all SOD was eluted as D, and with a decrease in the SOD concentration, the peak area of D decreased and concomitantly the monomers appeared. M2 could be the sole form in infinitely diluted SOD solution, and D was considered to be converted to M2 through M1. The SOD concentration giving 50% D ([SOD]1/2) was 1.0 mg/ml.(ABSTRACT TRUNCATED AT 250 WORDS)
Inouye
K
K
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University.
Osaki
A
A
Tonomura
B
B
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004912:Erythrocytes / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013482:Superoxide Dismutase / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D013696:Temperature
D013482:Superoxide Dismutase