No abstract

Electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) spectroscopies were used to characterize the binding of spin-labeled fatty acid (SLFA) to bovine serum albumin (BSA). Association constants of three stearic acid derivatives labeled with a nitroxyl radical at C-5, C-12, or C-16 were estimated by EPR spectroscopy as the ratio of SLFA to BSA was increased from about 0 to 9. The values were compared to those for unmodified stearate. With all three SLFA, it was apparent that the nitroxyl residue modified the binding pattern. For SLFA:BSA ratios up to 1, which probably involves the site(s) on BSA most specific for long-chain FA, the C-16 derivative bound with an affinity similar to that of the natural FA. At higher ratios, the association constants for this SLFA were lower than those for stearate. The C-12 and C-5 derivatives showed only low-affinity binding relative to stearate. The spectral parameter, W, was constant for SLFA:BSA ratios between 0 and 1 in the case of C-16 compound, indicating physical homogeneity of the high-affinity binding site. At higher ratios, the spectra changed progressively, indicating inhomogeneity of the lower affinity binding sites although parallel changes in association constants were not observed. Changes in W due to Heisenberg spin exchange were ruled out. By examining the mobility profile of the bound SLFA by both EPR and ST-EPR techniques, it was shown that the nitroxyl group was maximally immobilized when attached near the center of the carbon chain of the bound SLFA.

Saturation transfer electron paramagnetic resonance and the spin label 2(3-carboxypropyl)4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl were used to study erythrocytes from patients with Duchenne muscular dystrophy or Becker syndrome and from age-matched normal boys. There were significant differences in the spectral intensities of erythrocytes from Duchenne patients when compared to controls. Spectral intensities increased with time in the former; no such change was observed in the latter. Saturation transfer electron paramagnetic resonance spectra of erythrocytes from patients with Becker syndrome were significantly different from those from Duchenne patients but were not significantly different from normals. These observations suggest the possible usefulness of these techniques in the differential diagnosis of Duchenne muscular dystrophy. Spin label concentration spectral studies suggest that the observed spectral differences between Duchenne patients and controls were due to differential spin exchange phenomena.

Electron paramagnetic resonance (EPR) and saturation transfer–EPR (ST–EPR) techniques were employed to investigate the hydrodynamic properties of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Both apo- and holoenzyme were spin-labeled at the active site cysteine-149 residue with N- (1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) - maleimide. The apo- and holoenzymes were observed to have the same hydrodynamic structure and the spectroscopic results were consistent with these complexes behaving as spheres with hydrated radii of 41 Å. The environment of the paramagnetic electron was significantly more polar in the spin-labeled holoenzyme than in the spin-labeled apoenzyme, suggesting that either ionic residues are positioned closer to the active site in the holoenzyme or that ionic segments of coenzyme nicotinamide adenine dinucleotide (NAD+) itself may interact with the paramagnetic electron of the maleimide spin label. The dependence of the phase quadrature second harmonic absorption ST-EPR signal upon microwave and Zeeman modulation field intensities was examined with particular attention directed to effects of the variation of these fields over samples of finite dimensions. Such considerations are crucial when comparing data obtained employing different microwave resonators or different sample geometries within the same resonator. The problem of accurately establishing phase nulls for phase quadrature measurements was examined as was the problem of estimating phase resolution. Thus, the present work represents both a specific application and a generalization of the ST-EPR methodology proposed by Thomas, Dalton, and Hyde [J. Chem. Phys. 65, 3006 (1976)].