SUBUNIT ASSOCIATION AND SIDE-CHAIN REACTIVITIES OF BOVINE ERYTHROCYTE SUPEROXIDE-DISMUTASE IN DENATURING SOLVENTS

被引:80
作者
MALINOWSKI, DP [1 ]
FRIDOVICH, I [1 ]
机构
[1] DUKE UNIV, MED CTR, DEPT BIOCHEM, DURHAM, NC 27710 USA
关键词
D O I
10.1021/bi00590a005
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The copper- and zinc-containing superoxide dismutase of bovine erythrocytes retains its native molecular weight of 32 000 in 8.0 M urea for at least 72 h at 25 °C, as evidenced by sedimentation equilibrium analysis. Subsequent to prolonged exposure to urea, the dimeric enzyme could be dissociated by sodium dodecyl sulfate in the absence of reductants, indicating the absence of unnatural disulfide crosslinks. The sulfhydryl group of cysteine-6 was unreactive toward 5,5'-dithiobis(2-nitrobenzoic acid) or bromoacetic acid in both neutral buffer and 8.0 M urea. The histidine residues of the enzyme were resistant to carboxymethylation in neutral buffer and 8.0 M urea. However, when the enzyme was exposed to bromoacetic acid in the presence of 6.0 M guanidinium chloride and 1 mM (ethylenedinitrilo)tetraacetic acid (EDTA), both sulfhydryl and histidine alkylation were observed. Guanidinium chloride (6.0 M) increased the reactivity of the sulfhydryl group of cysteine-6 and allowed the oxidative formation of disulfide-bridged dimers. This was prevented by 1 mM EDTA. It follows that 8.0 M urea neither dissociates the native enzyme into subunits nor produces a conformation detectably different than that possessed under native conditions. © 1979, American Chemical Society. All rights reserved.
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页码:5055 / 5060
页数:6
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