RELAXATION-TIME DETERMINATIONS BY PROGRESSIVE SATURATION EPR - EFFECTS OF MOLECULAR-MOTION AND ZEEMAN MODULATION FOR SPIN LABELS

The EPR spectra of nitroxide spin labels have been simulated as a func tion of microwave field, H-1, taking into account both magnetic field modulation and molecular rotation. It is found that the saturation of the second integral, S, of the first harmonic in-phase absorption spec trum is approximated by that predicted for slow-passage conditions, th at is, S similar to H-1/root 1 + PH12, in all cases. This result is in dependent of the degree of inhomogeneous broadening. In general, the f itting parameter, P, depends not only on the T-1 and T-2 relaxation ti mes, but also on the rate of molecular reorientation and on the modula tion frequency. Calibrations for determining the relaxation times are established from the simulations. For a given modulation frequency and molecular reorientation rate, the parameter obtained by fitting the s aturation curves is given by 1/P = a + 1/gamma(e)(2)T(1) . T-2(eff), w here T-2(eff) is the effective T-2. For molecular reorientation freque ncies in the range 2 x 10(7)-2 x 10(8) s(-1), T-2(eff) is dominated by the molecular dynamics and is only weakly dependent on the intrinsic T-2(0), allowing a direct estimation of T-1. For reorientation frequen cies outside this range, the (T-1/T-2) product may be determined from the calibrations. The method is applied to determining relaxation time s for spin labels undergoing different rates of rotational reorientati on in a variety of environments, including those of biological relevan ce, and is verified experimentally by the relaxation rate enhancements induced by paramagnetic ions. (C) 1998 Academic Press.