Relaxation Time Determinations by Progressive Saturation EPR: Effects of Molecular Motion and Zeeman Modulation for Spin Labels

Abstract

The EPR spectra of nitroxide spin labels have been simulated as a function of microwave field,H1, 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 ∼ H1/1 +PH21, in all cases. This result is independent of the degree of inhomogeneous broadening. In general, the fitting parameter,P, depends not only on theT1andT2relaxation 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/γ2eT1·Teff2, whereTeff2is the effectiveT2. For molecular reorientation frequencies in the range 2 × 107–2 × 108s−1,Teff2is dominated by the molecular dynamics and is only weakly dependent on the intrinsicT02, allowing a direct estimation ofT1. For reorientation frequencies outside this range, the (T1T2) 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.