Comparative study of the interaction of CHAPS and Triton X-100 with the erythrocyte membrane.

The zwitterionic detergent CHAPS, a derivative of the bile salts, is widely used in membrane protein solubilization. It is a "facial" detergent, having a hydrophilic side and a hydrophobic back. The objective of this work is to characterize the interaction of CHAPS with a cell membrane. To this aim, erythrocytes were incubated with a wide range of detergent concentrations in order to determine CHAPS partition behavior, and its effects on membrane lipid order, hemolytic effects, and the solubilization of membrane phospholipids and cholesterol. The results were compared with those obtained with the nonionic detergent Triton X-100. It was found that CHAPS has a low affinity for the erythrocyte membrane (partition coefficient K=0.06mM(-1)), and at sub-hemolytic concentrations it causes little effect on membrane lipid order. CHAPS hemolysis and phospholipid solubilization are closely correlated. On the other side, binding of Triton X-100 disorders the membrane at all levels, and has independent mechanisms for hemolysis and solubilization. Differential behavior was observed in the solubilization of phospholipids and cholesterol. Thus, the detergent resistant membranes (DRM) obtained with the two detergents will have different composition. The behaviors of the two detergents are related to the differences in their molecular structures, suggesting that CHAPS does not penetrate the lipid bilayer but binds in a flat position on the erythrocyte surface, both in intact and cholesterol depleted erythrocytes. A relevant result for Triton X-100 is that hemolysis is not directly correlated with the solubilization of membrane lipids, as it is usually assumed.

Factors determining detergent resistance of erythrocyte membranes.

The degree of detergent insolubility of cell membranes is a useful parameter to test the strength of lipid-lipid interactions relative to lipid-detergent interactions. Thus, solubility studies could give insights about lipid-lipid interactions relevant in domain formation. In this work we perform a detailed study of the solubilization of four different erythrocyte membrane systems: intact human and bovine erythrocytes, and human and bovine erythrocytes depleted in cholesterol with methyl-beta-cyclodextrin. Each system was incubated with different concentrations of the non-ionic detergent Triton X-100, and the insoluble fraction was characterized by determining cholesterol and phosphorus content. A distinct solubilization behavior was obtained for the four systems, which was quantified by a "detergent resistance parameter" obtained from the fit of the solubility curves. In order to correlate these findings with membrane structural parameters, we quantify the degree of acyl chain order/rigidity of the original membranes by EPR spectroscopy, finding that detergent resistance is higher when acyl chains are more rigid. Regarding compositional properties, we found a good correlation between detergent resistance parameters and the total amount of cholesterol plus sphingomyelin in the original membranes. Our results suggest that a high degree of acyl chain packing is the determinant membrane factor for resistance to the action of Triton X-100 in erythrocytes.

Detergent solubilization of bovine erythrocytes. Comparison between the insoluble material and the intact membrane.

Early works have shown that when biomembranes are extracted with the non-ionic detergent Triton X-100 at 4 degrees C, only a subset of the components is solubilized. The aim of this paper was to investigate the solubilization of a cell membrane at different Triton concentrations, and to compare the lipid composition and acyl chain order/mobility of the insoluble material with those of the original membrane. We choose bovine erythrocytes, because they have an uncommon composition, as they have a huge amount of sphingomyelin and phosphatidylcholine is almost absent. We determined the degree of order/mobility of the lipid acyl chains by EPR spectroscopy, using liposoluble spin labels. Incubation of bovine erythrocytes with increasing Triton X-100 concentrations yields decreasing amounts of insoluble material which is enriched in sphingomyelin and depleted in cholesterol. Complete lipid solubilization is achieved at a detergent/lipid ratio of about 60, which is much higher than the values reported for human erythrocytes, but is in line with results obtained in model systems. An insoluble pellet is still obtained at higher Triton concentrations, which seems to consist mainly of protein. A very high correlation is found between lipid chain mobility restrictions and sphingomyelin content in the lipid structures. The human erythrocyte membrane also fits well in this correlation, suggesting a significant role of sphingomyelin in determining acyl chain organization. The analogies and differences between our insoluble material and the detergent-resistant membranes (DRM) are discussed.