Protein NMR spin trapping with [methyl-13C3]-MNP:: Application to the tyrosyl radical of equine myoglobin

被引:10
作者
Bose-Basu, B [1 ]
DeRose, EF [1 ]
Chen, YR [1 ]
Mason, RP [1 ]
London, RE [1 ]
机构
[1] NIEHS, Res Triangle Pk, NC 27709 USA
关键词
myoglobin; tyrosyl radical; spin trapping; MNP; NMR; free radicals;
D O I
10.1016/S0891-5849(01)00599-8
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
Direct spin trapping studies of protein radical adducts are limited as a consequence of the long rotational correlation times and consequent broadening of the ESR resonances. It can be difficult to determine both the nature and number of adduct species present. NMR detection of reduced spin adducts represents an alternate approach which, however, is subject to the limitations of lower sensitivity and a limited capability for isolating the resonances arising from the reduced adduct from other chemistry involving the spin trap. In the present study, we have utilized [methy1-C-13(3)]-MNP for the detection and analysis of tyrosyl spin adducts formed as a result of exposure of equine myoglobin to hydrogen peroxide. The methyl-C-13 label allows high detection sensitivity in two dimensions, narrow line widths and most significantly, removal by dialysis of unreacted spin trap as well as any nonprotein derivatives that may form. For equine myoglobin, it is found that adduct formation involves a single residue-Tyr-103 and further that adduct formation occurs at the C-3 carbon of the amino acid. HMQC-NOESY experiments further revealed the proximity of the labeled methyl groups to both the three aromatic tyrosyl protons as well as the aromatic protons of the nearby Phe-106 residue. (C) 2001 Elsevier Science Inc.
引用
收藏
页码:383 / 390
页数:8
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