Vertical fluctuations of the terminal methyl groups of stearic acid acyl chains toward the surface of dimyristoylphosphatidylcholine (DMPC) bilayers have been investigated by using spin-label electron-electron double-resonance ( ELDOR ) methodology. Spin-label pairs consisting of two populations of stearic acid spin-labels were employed, each at 0.25 mol% concentration, where the nitroxides of the first population were 15N substituted and the nitroxides of the second contained 14N. Various combinations of labels with the nitroxide moieties located at carbons 5, 12, or 16 (C5, C12, C16) were used. ELDOR permits measurement of collision frequencies between the two constituents of the pair, for example, between 15N spin-labels at C5 and 14N labels at C16. Intramolecular contributions to the ELDOR effect including nitrogen nuclear relaxation are eliminated by the use of spin-label pairs. Above the main phase transition temperature, bimolecular collisions between C5 and C16 occur with about half the frequency of C16:C16 collisions. It is concluded that vertical fluctuations are very pronounced. A dependence of these fluctuations on temperature and pH has been observed. Lateral diffusion constants calculated from the bimolecular collision frequencies of C16:C16 pairs are 4.56 X 10(-8), 5.77 X 10(-8), and 8.09 X 10(-8) cm2/s at 27, 37, and 47 degrees C. These values are in good agreement with previous measurements of lipid diffusion in DMPC.

We have developed a new membrane-impermeant, bifunctional spin-labeling reagent, bis-(sulfo-N-succinimidyl) doxyl-2-spiro-4'-pimelate (BSSDP), and employed it in an electron paramagnetic resonance (EPR) study of the rotational diffusion of the anion-exchange channel (band 3) in intact human erythrocytes. BSSDP reacts in a covalent manner and with high specificity with the extracytoplasmic domain of band 3, forming a complex in which the spin-label is immobilized on the protein. The linear EPR spectrum of BSSDP-labeled intact erythrocytes is characteristic of a highly immobilized, spatially isolated nitroxide probe. The saturation-transfer EPR spectrum of the same sample indicates that the anion channel in intact erythrocytes exhibits rotational dynamics in the 0.1-1 ms correlation time range at 20 degrees C. Rotational dynamics in this motional domain are consistent with a strong interaction of the anion-exchange channel with the erythrocyte cytoskeleton. The saturation-transfer EPR spectrum of ghosts prepared from BSSDP-labeled erythrocytes indicates a significant increase in rotational mobility of the anion channel, suggesting a significant disruption on lysis of interactions between the anion channel and the cytoskeleton.

The resolution and sensitivity of electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) for biological applications are greatly improved by deuteration and substitution of 15N for 14N in the spin-labeled probe N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (MSL We have shown that the sensitivity and resolution of electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) spectroscopy were substantially improved for biological studies by deuteration of the frequently used maleimide spin label N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (MSL) which binds covalently to proteins (1, 2). Significant gains in detectability and resolution were observed with [2H]MSL in both the fast motion limit for freely tumbling spin label (rc 10 psec) and in the slow to very slow correlation time range exhibited by labeled proteins such as bovine serum albumin and hemoglobin in viscous media (0.1 ,usec s rT ' 1 msec). The improvements resulted from a reduction in the inhomogeneous line broadening due to the weaker superhyperfine interactions of the unpaired electron with deuterium than with hydrogen.To further advance our capabilities for interpretation of spectra of labeled proteins, we prepared the doubly substituted that 15N or deuterium substitution increased sensitivity for a freely tumbling spin label and that '5N simplified the spectrum by reduction of the number of nuclear manifolds from three to two (4-9). However, a doubly substituted probe with '5N and deuterium had not been prepared previously. The potential increase in sensitivity and resolution of such a probe seemed particularly advantageous in biological systems in which the substances to be spin labeled are often present in low concentrations and are macromolecules with slow correlation times in the range appropriate for analysis by ST-EPR techniques. (11,12) and spin labeled with a given maleimide analogue at a ratio of one spin label per tetramer as described by Beth et al. (13). GAPDHase activity was 28% inhibited by MSL and its two analogues.

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