Surface behavior of detergent-resistant membrane monolayers from human erythrocyte

CLEYTON C. DOMINGUES; EDUARDO M. CLOP; MARÍA A. PERILLO; ENEIDA DE PAULA

Los Cocos, Córdoba, Argentina

Congreso; XXXVIII Reunión de la Sociedad Argentina de Biofísica; 2009

Sociedad Argentina de Biofísica

The detergent-resistant membrane (DRM) fractions are obtained after treatment with nonionic detergents at 4°C. We have previously shown, by means of electron paramagnetic resonance (EPR), that DRMs from human erythrocytes prepared with Triton X-100 at 4°C present an increased molecular organization in comparison to intact membranes, as expected from the liquid ordered state of DRMs. In this study DRMs were isolated by ultracentrifugation of detergent-treated whole RBCs in sucrose density gradients. The Gibbs adsorption isotherm of DRMs, Ghost membranes as well as their extracted lipids at the air-water interface were analysed by evaluating the time-dependent variation of the lateral surface pressure (Pi ) after injecting the membrane in the subphase. DRMs resulted unable tospread at the air-water interface. This reflected their close molecular packing and was in in agreementwith our EPR study. On the contrary Ghost membranes as well as their extracted lipids could spread to form stable monolayers. Lipids extracted from DRMs were also spread directly from their chloroform-methanol (2:1) solution onto the air-water interface and the variation of  as a function of the trough area was recorded to obtain the Pi-A compression isotherms. A well-defined collapse point was detected at 50 mN/m for Ghost monolayers while monolayers of lipids extracted from ghosts and from DRMs collapsed at ca. 46 mN/m. At a Pi of ca. 25 mN/m the isotherm of the DRM lipids monolayer exhibited a bidimensional phase transition with a significant change in its compressibility. In DRM lipids, microscopy (epifluorescence) Pi =10 mN/m revealed the coexistence different liquid condensed domains that tended to coalesce at higher Pi. In DRM lipids above the phase transition point as well as monolayers from ghost extracted lipids the BAM images were dominated by bright dots possibly reflecting the absence of a pure monolayer phase.