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 function 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 spectrum is approximated by that predicted for slow-passage conditions, that is, S similar to H-1/root 1 + PH12, in all cases. This result is independent of the degree of inhomogeneous broadening. In general, the fitting parameter, P, depends not only on the T-1 and T-2 relaxation times, but also on the rate of molecular reorientation and on the modulation 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 saturation curves is given by 1/P = a + 1/gamma(e)(2)T(1) . T-2(eff), where T-2(eff) is the effective T-2. For molecular reorientation frequencies 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 frequencies outside this range, the (T-1/T-2) product may be determined from the calibrations. The method is applied to determining relaxation times for spin labels undergoing different rates of rotational reorientation in a variety of environments, including those of biological relevance, and is verified experimentally by the relaxation rate enhancements induced by paramagnetic ions. (C) 1998 Academic Press.