Protein NMR spin trapping with [methyl-13C(3)]-MNP: application to the tyrosyl radical of equine myoglobin.

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 [methyl-13C(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-13C 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.

Deuterium nuclear spin-lattice relaxation times and quadrupolar coupling constants in isotopically labeled saccharides.

(13)C and (2)H spin-lattice relaxation times have been determined by inversion recovery in a range of site-specific (13)C- and (2)H-labeled saccharides under identical solution conditions, and the data were used to calculate deuterium nuclear quadrupolar coupling constants ((2)H NQCC) at specific sites within cyclic and acyclic forms in solution. (13)C T(1) values ranged from approximately 0.6 to 8.2 s, and (2)H T(1) values ranged from approximately 79 to 450 ms, depending on molecular structure (0.4 M sugar in 5 mM EDTA (disodium salt) in (2)H(2)O-depleted H(2)O, pH 4. 8, 30 degrees C). In addition to providing new information on (13)C and (2)H relaxation behavior of saccharides in solution, the resulting (2)H1 NQCC values reveal a dependency on anomeric configuration within aldopyranose rings, whereas (2)H NQCC values at other ring sites appear less sensitive to configuration at C1. In contrast, (2)H NQCC values at both anomeric and nonanomeric sites within aldofuranose rings appear to be influenced by anomeric configuration. These experimental observations were confirmed by density functional theory (DFT) calculations of (2)H NQCC values in model aldopyranosyl and aldofuranosyl rings.