DEPENDENCE OF THE MULTIPLE-QUANTUM EPR SIGNAL ON THE SPIN-LATTICE RELAXATION-TIME - EFFECT OF OXYGEN IN SPIN-LABELED MEMBRANES

Methods to detect intermodulation sidebands produced upon irradiation of an EPR spin system by two closely spaced microwave frequencies (separation ω1-ω2) were described by Sczaniecki et al. (J. Chem. Phys.94, 5907, 1991). The first pair of sidebands correspond to absorption of two quanta from one source and emission of one quantum from the other source and are therefore designated as three-quantum transitions. Similarly, the second pair of sidebands are five-quantum transitions. At low incident power, it is shown that the signal intensities of three-quantum spectra of spin labels are proportional to the spin-lattice relaxation time T1, using [O2] as a convenient T1 modulator. The ratio of three-to five-quantum signal intensities also is proportional to T1. Signal intensities were studied as a function of the separation of the two irradiating frequencies. The three-quantum intensity drops to 1 2 at (ω1 - ω2)T1 ≃ 1, which was confirmed by pulse experiments and also by varying the frequency difference and [O2]. Hydrophobic and hydrophilic components of the spin label Tempo in equilibrium with a lipid bilayer were separated by subtraction of three-quantum spectra obtained in the presence and absence of O2. Multiple-quantum EPR is found to be a useful tool in spin-label oximetry, in discrimination of overlapping spectra with differing T1 values, and in estimating numerical values of T1. © 1993 Academic Press. All rights reserved.