Hemin-induced lipid membrane disorder and increased permeability: a molecular model for the mechanism of cell lysis

Abstract

The interaction between hemin and lipid bilayers was examined from the point of view of: (a) changes in membrane permeability, (b) changes in lipid organization, (c) catalysis of lipid peroxidation. The leakage of solutes trapped in inner aqueous compartments of vesicles and liposomes increased in a concentration-dependent manner, in the range of 100-1000 microM, saturating at ca. 400 microM hemin. Under saturating conditions, leakage was essentially complete after ca. 0.5 h. Antioxidants had practically no effect upon this behavior. Extensive leakage at 5 and 10 microM hemin was observed only in the absence of antioxidants and after ca. 10 h. Electron spin resonance spectra of a spin label incorporated in oriented lipid multibilayers indicated that hemin decreased the degree of order of the bilayer. The effect was instantaneous, time-independent, and unaffected by antioxidants, displaying a concentration dependence similar to that of the permeability studies. In contrast, hemin-catalyzed lipid peroxidation displayed a bell-shaped dependence on hemin concentration, the effect ceasing at concentrations lower than those required for instantaneous permeability and structural changes. The bell-shaped behavior was found both for the detection of thiobarbituric acid-reactive compounds (TBARS) and for oxygen consumption. The time scale of TBARS formation was much slower than that for permeability changes, significant amounts of products being detected only after leakage was essentially complete. Both permeability and lipid peroxidation occurred to a larger extent in cholesterol-containing membranes. Hemin is highly aggregated in aqueous phase. Upon binding to the membrane, the aggregates undergo dissociation in a hemin/lipid ratio-dependent manner. At low ratios, hemin is largely monomeric, while at high ratios, aggregates prevail. Monomers are more effective in promoting oxidation. Aggregates are responsible for the enhancement of permeability and membrane disorder. Oxidation of membrane components is often proposed to be involved in the mechanism of hemin-promoted cell lysis. Nevertheless, the role of oxidative processes is not clear in some hemoglobinopathies. The present results provide a molecular model for an alternative mechanism of lysis that would dominate in cases where the concentration is high enough for hemin to aggregate.