Vol. 152, No. 1, 1988 April 15, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 344-350 USE OF MEMBRANE SPIN LABEL SPECTRA TO MONITOR RATES OF REACTION OF PARTITIONING COMPOUNDS: HYDROLYSIS OF A LOCAL ANESTHETIC ANALOG M. Lucia Bianconi I, Antonia T. ido Amaral 2 and Shirley Schreier Departments of Biochemistry I and Chemistry 2 Instituto de Qufmica, Universidade de S~o Paulo C.P. 20780, CEP 01498, S. Paulo, Brazil Received February 25, 1988 ESR spectra of membrane spin probes are conventionally used to obtain structural information. Here we show, for the first time, that when a membrane-soluble compound undergoes a chemical reaction, time-dependent changes in the ESR spectra of membrane spin probes can yield information about the kinetics of reaction. A benzoic acid ester, analog of the local anesthetic tetracaine, partitions between aqueous and membrane phases, causing changes in membrane structure as monitored by the ESR spectra of a probe. At alkaline pH, the lineshapes are time-dependent and the spectra go back to that in the absence of drug. The changes follow pseudo-first order kinetics. This effect is due to drug hydrolysis leading to water-soluble products, as confirmed by direct spectrophotometric measurements of the reaction. The pseudo-first order rate constants found by the latter method are in very good agreement with those calculated by ESR. The rate of hydrolysis decreases with increasing membrane concentration. This phenomenon accounts in part for the increased potency and toxicity of the more membrane- soluble local anesthetics. ©1988AcademicPress, Inc. ESR spectra of membrane spin labels are conventionally employed to analyse structural properties of membrane components (usually lipids) (I). A large body of work has been done examining the effects caused by membrane-soluble pharmacologically active drugs upon the organization and dynamics of biological membranes and lipid bilayers. Among these, local anesthetics have been widely examined (2-8). In conjunction with other techniques, in particular deuterium NMR (9-15), it has been found that the charged and uncharged forms of local anesthetics partition into membranes to a 0006-291X/88 $1.50 Copyright © 1988~ Aca~mic Press, ~c. AHr~h~ ~repro~ctionina~rm reserved. 344