NEW EPR METHODS FOR THE STUDY OF VERY SLOW MOTION: APPLICATION TO SPIN‐LABELED HEMOGLOBIN

Nitroxide-radical spin labels have been widely used to study motion in biological systems. Smith l has recently reviewed the subject and provides access to the literature. The present work is concerned with the development of methodology that will permit studying slower motions than has previously been possible. Practically, the spin-label technique has been restricted to rotational correlation times T~ shorter than 3 x lo-; set." Theoretically, any motion affects the ordinary linear epr spectrum, but several problems arise as the motion becomes slow. ( 1 ) The effect of motion on the spectrum becomes less and less as the spectra asymptotically approach the line shape expected from a rigid powder. Hence, the signal-to-noise ratio of tne motional effects goes to zero. (2) Kreilick has observed temperature-dependent couplings of nitroxide radicals using nmr techniques. Thus, the inhomogeneous line width (i.e., the line width that would be observed in a single crystal, and arising from unresolved hyperfine couplings to protons of the radical) is temperature-dependent, and this affects the spectral shape. (3 ) It is necessary to know the theoretical line shape that is approached as the motion slows, but the expediency of freezing the sample fails because the dielectric constant of the solvent affects the hyperfine coupling and is itself generally temperature-dependent and changes markedly upon freezing. It has occurred to us that methods involving the nonlinear response of the spin system in which intense irradiating fields are employed and the observed spectrum depends to a considerable degree on relaxation to the lattice might permit determination of very slow motion. If the spin-state lifetime limiting line width is l/yTl,,, then the slowest motion that could in principle be measured by a nonlinear method would involve spectral diffusion by an amount I/yT,,. in a time T,,,. Here, y is the gyromagnetic ratio of the electron and T,,, is the electron longitudinal (or spin-lattice) relaxation time. Assuming that the anisotropy, AW. of the typical nitroxide radical is about 2s X loy rad (this being the spectral width swept out by the m, = -1 nuclear spin configuration as the radical undergoes isotropic rotational diffusion) and that TI, = 1 W sec, we can estimate a limiting maximum T : : T ? = T , < , ( A W T ~ , . ) ~ 0 4 sec. Success in extending the present range to times as long as 1W3 or lo--’ sec would be very useful. These slow times are relevant for many biological processes, and this is a time scale not readily accessible to other analytical methods.