Lipid chain ordering in detergent resistant erythrocyte membrane domains studied by spin label EPR. Comparison of results obtained with Triton X-100 and CHAPS.

RODI P; VELÁZQUEZ M; GENNARO AM

Praia Vermelha - Rio de Janeiro - Brasil

Workshop; 2º International Workshop on Spectroscopy for Biology; 2004

Washed erythrocytes, either intact or cholesterol-depleted, were incubated with the non ionic detergent Triton X-100 or with the zwitterionic detergent CHAPS in ice. The pellets containing the detergent resistant membrane domains (DRM) were washed several times and labeled with 5-, 12-, and 16-SASL (stearic acid spin labels). An interest result is that while cholesterol-depleted erythrocytes were lysed by CHAPS, a very low hemolysis degree was achieved with intact cells. EPR spectra were recorded at room temperature and the apparent order parameter S was calculated. The “fluidity gradient” showed differences among the different cases and with the intact membrane (IM). As instance, at the level of the 5th and 12th carbons, we obtained SCHAPS > STRITON > SIM, indicating straighter acyl chains in DRM isolated with CHAPS. The analysis of lipid composition by thin layer chromatography also showed differences. The fact of finding differences between DRM obtained with different detergents reinforce the hypothesis that DRM composition does not necessarily reflect that of the rafts in the original cell membrane. Thus, caution should be exercised when interpreting the biological significance of experiments on DRM.