Multiple stages of detergent-erythrocyte membrane interaction—A spin label study

Abstract

The various stages of the interaction between the detergent Triton X-100 (TTX-100) and membranes of whole red blood cells (RBC) were investigated in a broad range of detergent concentrations. The interaction was monitored by RBC hemolysis-assessed by release of intracellular hemoglobin (Hb) and inorganic phosphate-and by analysis of EPR spectra of a fatty acid spin probe intercalated in whole RBC suspensions, as well as pellets and supernatants obtained upon centrifugation of detergent-treated cells. Hemolysis finished at ca. 0.9mM TTX-100. Spectral analysis and calculation of order parameters (S) indicated that a complex sequence of events takes place, and allowed the characterization of various structures formed in the different stages of detergent-membrane interaction. Upon reaching the end of cell lysis, essentially no pellet was detected, the remaining EPR signal being found almost entirely in the supernatants. Calculated order parameters revealed that whole RBC suspensions, pellets, and supernatants possessed a similar degree of molecular packing, which decreased to a small extent up to 2.5mM detergent. Between 3.2 and 10mM TTX-100, a steep decrease in S was observed for both whole RBC suspensions and supernatants. Above 10mM detergent, S decreased in a less pronounced manner and the EPR spectra approached that of pure TTX-100 micelles. The data were interpreted in terms of the following events: at the lower detergent concentrations, an increase in membrane permeability occurs; the end of hemolysis coincides with the lack of pellet upon centrifugation. Up to 2.5mM TTX-100 the supernatants consist of a (very likely) heterogeneous population of membrane fragments with molecular packing similar to that of whole cells. As the detergent concentration increases, mixed micelles are formed containing lipid and/or protein, approaching the packing found in pure TTX-100 micelles. This analysis is in agreement with the models proposed by Lasch (Biochim. Biophys Acta 1241 (1995) 269-292) and by Le Maire and coworkers (Biochim. Biophys. Acta 1508 (2000) 86-111).

Biophysical approaches in the study of biomembrane solubilization: quantitative assessment and the role of lateral inhomogeneity

“…We have proposed that hemoglobin is released from erythrocytes through pores created by the partition of TX-100 (Preté et al 2011). Likewise, Gennaro and colleagues.…”

“…Among the spectroscopic techniques used in the study of detergent-membrane interactions, electron paramagnetic resonance (EPR) has been one of the most important, since one can monitor molecular order and dynamics in both bilayers and micelles (Preté et al 2011). EPR spectra taken from such membrane systems-doped with spin labels-are sensitive to changes in molecular organization caused by the partitioning of amphiphiles, phase separation, bilayer/micelle transition, etc.…”

“…EPR spectra taken from such membrane systems-doped with spin labels-are sensitive to changes in molecular organization caused by the partitioning of amphiphiles, phase separation, bilayer/micelle transition, etc. For instance, detergent incorporation into erythrocytes decreases the molecular order and increases the mobility of the phospholipid hydrocarbon chains (Galembeck et al 1998;Preté et al 2011;Rodi et al 2006Rodi et al , 2014 and induces phase separation (Crepaldi Domingues et al 2009;Domingues et al 2010;Preté et al 2011;Rodi et al 2006Rodi et al , 2014Casadei et al 2014a). …”

“…First, EPR has been used to determine the order parameter of TX-100 micelles (S = 0.46). Then, the hemolytic effect of Triton X-100-followed either by inorganic phosphate or Hb release-was determined at high lipid contents (Ht 10-50%), and C sat and C sol values were found in the range of 0.5-0.9 mM TX-100 (Preté et al 2011). Finally, EPR spectra of whole blood, supernatant and pellet fractions of the hemolytic experiment were obtained, revealing important details of the hemolytic effect: (1) the order parameter of erythrocyte membranes (S = 0.710) slightly decreased at TX-100 concentrations higher than C sat , and an anisotropic spectrum was detected in the supernatant, which S values were comparable to those of erythrocyte membranes; (2) at TX-100 concentrations higher than C sol the S values in the supernatant steadily decreased, and (3) they reach micelle-like values (S < 0.5) at excess (10 times C sol ) TX-100.…”

“…Biophysicists are particularly interested in the physicochemical and molecular aspects of detergent-membrane interaction, such as the interplay between bilayer and micellar structure as a function of lipid/detergent molar ratio (e.g., Kragh-Hansen et al 1998;Heerklotz and Seelig 2000;Preté et al 2011;Lichtenberg et al 2013). …”