Biomedical EPR Part-B Methodology Instrumentation and Dynamics - Sandra R. Eaton

302 CANDICE S. KLUG AND JIMMY B. FEIX

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unremarkable and an essential development of the orientationally resolved method was the introduction of non-linear spectra detected in quadrature phase with the static field modulation (Hyde and Dalton, 1972). Such out-of- phase spectra have appreciable intensity only in the presence of saturation. The result is that the lineshapes of certain of these non-linear displays are exquisitely sensitive to rotational motion on the

Figure 1. Schematic indication of saturation transfer processes (arrows) in axial powder spectra (heavy lines) for two ensembles of spins, b and f (hatched). Within ensemble b, saturation transfer takes place between orientationally selected component spin packets, i and j (grey), by internal exchange processes: either rotational diffusion or Heisenberg spin exchange Two-site exchange leads to transfer of saturation between spin ensembles b and f, with characteristic rate constants and

ST-EPR has come conventionally to be identified with studies of slow rotational motion. It is clear, indeed already to the originators, that this class of experiment can be used to study any type of slow molecular motion that gives rise to transfer of saturation particularly, for instance, two-site exchange (see Fig. 1). Further, the methods are applicable also to the quantitation of any magnetic interaction that alleviates saturation. Especially prominent among these are Heisenberg spin-exchange between nitroxides and interactions with paramagnetic relaxants such as transition metal complexes or molecular oxygen. The latter has been studied intensively by Hyde and coworkers, principally by saturation recovery EPR methods (Hyde and Subczynski, 1989). enhancement currently constitutes the most powerful EPR methodology in site-directed spin-labelling applications (Hubbell and Altenbach, 1994). The advantage of saturation-based methods is that they are sensitive to much weaker interactions than are the

conventional linewidths which are determined by the rate rather than by the slower rate.

Here we aim to cover ST-EPR in its widest sense. Appropriate to the dedication of this volume, we begin with a description of the historical development of the subject. At its baldest this simply can be stated as: “nothing would have happened without Jim Hyde”. What follows is then devoted to a description of ST-spectroscopy up to its state-of-the-art application. Initial emphasis is placed on rotational diffusion measurements, but in-depth coverage is given also to the less conventional applications for studying exchange processes and paramagnetic relaxation enhancements. The advantage of integral methods is stressed throughout because they are generally insensitive to inhomogeneous broadening and the intensities are additive in multi-component systems.

2.HISTORICAL DEVELOPMENT

Saturation transfer spectroscopy has its origin in the observation by Hyde and collaborators (1970) that the CW saturation behaviour of the conventional low-temperature EPR spectra from flavin free radicals depended on temperature in a way compatible with slow molecular rotation. Following the classic analysis of adiabatic rapid-passage non-linear EPR spectra by Portis (1955) and Weger (1960), Hyde and Dalton (1972) explored the sensitivity to slow rotational diffusion of the first harmonic dispersion spectrum detected 90°-out-of-phase with respect to the field modulation. In the rigid limit, this should have the pure (zeroth harmonic) absorption lineshape, Rotation-dependent saturation transfer causes angularly selective decreases in intensity of the ST-EPR lineshape, relative to the absorption spectrum (Fajer and Marsh, 1983a). The predicted sensitivity of the to slow motion was found for a small nitroxide spin label in supercooled glasses (Hyde and Dalton, 1972). Additionally, the ST-EPR spectra were found to depend on the fieldmodulation frequency, in a manner expected for rapid passage conditions. Essentially, the is determined by the product where is the rotational correlation time. (Throughout this chapter, harmonics refer to a Fourier expansion of the EPR signal with respect to Experimentally, the various harmonic spectra are obtained by phasesensitive detection at frequencies )

At the time, dispersion spectra were thought to be unsuitable for biological applications because of problems associated with sensitivity and klystron FM noise. A systematic search was therefore conducted by Hyde and Thomas (1973) to identify the out-of-phase ST-EPR display best suited