EPR STUDY OF MEMBRANE PARTITIONING, ORIENTATION, AND MEMBRANE-MODULATED ALKALINE-HYDROLYSIS OF A SPIN-LABELED BENZOIC-ACID ESTER

The composite EPR spectra of a spin probe (Tempobenzoate, TB) that partitions between aqueous and egg phosphatidylcholine (EPC) membrane phases provide information about (1) its location and orientation in the membrane, (2) its partition coefficient and the thermodynamics of the process, and (3) the kinetics of its alkaline hydrolysis, a novel application of spin label spectra. The broad and narrow spectral components of the probe in the membrane and aqueous phase, respectively, are sufficiently resolved in the high-field region, to permit quantitative evaluation of the narrow line (h). The line shapes of membrane spectra, obtained by spectral subtraction, indicate that the nitroxide x axis (the probe long molecular axis) is aligned preferentially parallel to the bilayer normal. Spectral subtractions, as well as measurement of h, are used to calculate the probe's partition coefficient. Thermodynamic analysis indicated that binding of the probe is endothermic and driven by an increase in entropy, suggesting that dehydration is the predominant step. At alkaline pH, TB undergoes hydrolysis. Although the narrow high-field lines of both reactant (TB) and product (Tempol) overlap, the pseudo-first-order rate constants can be calculated from the time-dependent height of this line. At high ionic strength, the reaction occurs essentially in the aqueous phase, and the decrease in rate with increasing membrane concentration is due to partition modulation of reagent concentration. TB-membrane binding was also evaluated from the kinetic data. The results are in excellent agreement with those obtained by direct measurements.