Rotational diffusion studied by passage saturation transfer electron paramagnetic resonance

A comprehensive description is given of instrumental and theoretical methods employed to make accurate measurements of rotational correlation times using passage saturation transfer electron paramagnetic resonance (ST-EPR). Saturation transfer methods extend by several orders of magnitude the sensitivity of EPR to very slow motion; for example, for nitroxide spin labels, correlation times as long as 10^-3 sec become accessible to measurement. Two ST-EPR detection schemes are discussed in detail: dispersion, detected 90° out of phase with respect to the 100 kHz field modulation, and absorption, detected 90° out of phase with respect to the second harmonic of the 50 kHz field modulation. The sensitivities of these configurations are illustrated with experimental spectra obtained from a system obeying isotropic Brownian rotational diffusion; namely, maleimide spin labeled human oxyhemoglobin in aqueous glycerol solutions. Two theoretical approaches, one employing coupled Bloch equations and the other utilizing the stochastic Liouville equation for the density matrix with the orientation variables treated by transition rate matrix or orthogonal eigenfunction expansion methods, are in excellent agreement with each other and with model system spectra. Both experimental and theoretical spectra depend on a number of relaxation processes other than rotational diffusion; consequently, considerable care must be taken to ensure the accuracy of measured rotational correlation times. Although the absorption method is currently the more sensitive and convenient one to apply with most conventional (commercial) spectrometers, the dispersion ST-EPR method is potentially more powerful, providing strong motivation for future technological efforts to decrease noise levels in dispersion experiments.