ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 336, No. 1, December 1, pp. 157–162, 1996 Article No. 0544 Physicochemical Characterization of Plasma Membranes from Density-Separated Trout Erythrocytes Rosita Gabbianelli,* Anna Maria Santroni,* Giancarlo Falcioni,* Enrico Bertoli,† Giovanna Curatola,† and Giovanna Zolese† ,1 *Dipartimento Biologia, MCA, Universita ` di Camerino, Camerino, Italy; and †Istituto di Biochimica, Facolta ` di Medicina e Chirurgia, Universita ` di Ancona, via Ranieri, 60131 Ancona, Italy Received June 11, 1996, and in revised form September 10, 1996 brane cholesterol, and phospholipid content (4, 5). Erythrocytes of Salmo irideus trout were separated Moreover, because of its role in oxygen transport, the in the range from 45 to 65% Percoll, yelding three well- erythrocyte is exposed to oxidative damage which could separated different fractions. Steady-state fluores- be related to significant lipid peroxidation in older cells cence of probes embedded in erythrocyte membranes (1). Human RBCs may be separated in three/five popu- and/or in liposomes from extracted lipids was used to lations by discontinuous density gradients (2, 6), since characterize their physicochemical properties. Fur- older cells are characterized by increasing density. Us- thermore, the fluorescence decay of 1,6-diphenyl-1,3,5- ing a similar technique it is possible to separate the hexatriene (DPH), embedded in the same liposomes, nucleated Salmo irideus trout erythrocytes in the was measured by a frequency decay fluorometer. DPH range from 45 to 65% Percoll, obtaining three well- decay was analyzed on the assumption of continuous separated fractions. Previous studies demonstrated distribution of lifetimes, for evaluating modifications marked differences between human (anucleate) and of membrane microheterogeneity. Significant differ- trout (nucleated) density-separated erythrocytes (7), ences were observed in the parameters measured for e.g., in catalase and glutathione peroxidase activities the three erythrocyte fractions, possibly connected which increase from the top to the bottom fraction, with the specific lipid composition of the samples. suggesting an increased protection against oxidation  1996 Academic Press, Inc. in dense cells of this aquatic organism. The aim of the Key Words: trout erythrocytes; density-separated present work was to characterize the physicochemical fractions; Laurdan; DPH; time-resolved fluorescence; properties of plasma membranes from density-sepa- microheterogeneity. rated trout erythrocytes. Such data could be of interest for a better understanding of the molecular mecha- nisms involved in the adaptation of aquatic organisms to particular living conditions. Our studies were car- The senescence process in human red blood cells ried out by steady-state and time-resolved fluorescence (RBCs) 2 is characterized by biochemical and physical using two different probes located in different parts of changes, extensively studied in previous works (1 – 3). the lipid bilayer. Fluorescence spectroscopy has been During their life span RBCs undergo a series of bio- largely used to study membrane characteristics, such chemical and physical changes involving both cytosolic as fluidity and polarity. Moreover, this technique has and membrane molecular structures. These age-re- been recently proposed as a powerful tool for investi- lated modifications are due to various biochemical pro- gating membrane heterogeneity (8, 9). Composition cesses, such as oxidation, proteolysis, and glycation, changes of membranes were correlated with fluores- and they are evident also at the membrane level, show- cence data obtained from cells sampled in different pe- ing, among others, modifications of fluidity (2), mem- riods of the year, to evaluate the possibility of adapta- tion of the fish environmental conditions. 1 To whom correspondence should be addressed. Fax: /39 71 2204398. MATERIALS AND METHODS 2 Abbreviations used: RBCs, red blood cells; DPH, 1,6-diphenyl- 1,3,5-hexatriene; POPOP, 2,2-p-phenylene-bis(5-phenyl)oxazole; All reagents were of analytic grade. Percoll was obtained from Sigma; 2-dimethylamino(6-lauroyl)naphthalene (Laurdan) and PC, phosphatidylcholine; FWHM, full width at half maximum. 157 0003-9861/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.