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 = 0.06 mM^− 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.     

Integral membrane protein interaction with Triton cytoskeletons of erythrocytes

The organization of erythrocyte membrane lipids and proteins has been studied following the release of cytoplasmic components with the non-ionic detergent Triton X-100. After detergent extraction, a detergent-resistant complex called the erythrocyte cytoskeleton is separated from detergent, solubilized lipid and protein by sucrose buoyant density sedimentation. In cytoskeletons prepared under isotonic conditions all of the major erythrocyte membrane proteins are retained except for the integral protein, glycophorin, which is quantitatively solubilized and another integral glycoprotein, band 3, which is only 60% removed. When cytoskeletons are prepared in hypertonic KCl solutions, band 3 is fully solubilized along with bands 2.1 and 4.2 and several minor components. The resulting cytoskeletons have the same morphology as those prepared in isotonic buffer but they are composed of only three major peripheral proteins, spectrin, actin and band 4.1. We have designated this peripheral protein complex the 'shell' of the erythrocyte membrane, and have shown that the attachment of band 3 to the shell satisfies the criteria for a specific interaction. Although Triton did affect erythrocyte shape, cytoskeleton lipid content and the activity of membrane proteases, there was no indication that Triton altered the attachment of band 3 to the shell. We suggest that band 3 attaches to the shell as part of a ternary complex of bands 2.1, 3 and 4.2.     

Mode of interaction of polyoxyethyleneglycol detergents with membrane proteins

Binding of dodecyloctaethyleneglycol monoether (C12E3) and purified Triton X-100 to various integral membrane proteins was studied by chromatographic procedures. Binding capacity decreased in the following order: bovine rhodopsin greater than photochemical reaction center greater than sarcoplasmic reticulum Ca2+-ATPase. The detergents were bound in different amounts to the proteins and less than corresponding to the aggregation number of the pure micelles. Appreciable binding of C12E8 to Ca2+-ATPase was observed far below the critical micelle concentration, consistent with interaction of the membrane protein with non-micellar detergent. Model calculations indicate that the detergents cannot combine with the membrane proteins, forming an oblate ring similar to that of pure detergent micelles, such as has been previously proposed for e.g. cytochrome b5 [Robinson and Tanford (1975) Biochemistry, 14, 365-378]. Other arrangements (prolate and monolayer rings), in which all detergent molecules are in contact with the protein, are considered as alternatives for covering the hydrophobic surface of the membrane protein with a continuous layer of detergent.     

Mode of interaction of phosphofructokinase with the erythrocyte membrane

Phosphofructokinase is known to associate with the human erythrocyte membrane both in vitro and in vivo. Such association activates the enzyme in vitro by relieving the allosteric inhibition imposed by ATP (Karadsheh, N.S., and Uyeda, K. (1977) J. Biol. Chem. 252, 7418-7420). We now demonstrate that ADP, ATP, and NADH, all of which are known to bind to the enzyme's adenine nucleotide activation site, are particularly potent in eluting the enzyme from the membrane. In addition, both inside-out red cell membrane vesicles and a 23-kDa fragment containing the amino terminus of the membrane protein, band 3, cause a slow, partial, and reversible inactivation of phosphofructokinase. The dependence of the residual phosphofructokinase activity on phosphofructokinase concentration demonstrates that inactivation occurs through the dissociation of active tetramers to inactive dimers. Dimers of phosphofructokinase associate with the membrane more avidly than tetramers. The kinetics of phosphofructokinase inactivation are consistent with the dissociation of tetramers in solution followed by the binding of dimers to the membrane. There is no indication of an association equilibrium between tetramers and dimers of phosphofructokinase bound to the membrane. Taken together, these results suggest that the amino-terminal segment of band 3 binds to the adenine nucleotide activation site, which is thought to be located in a cleft between the dimeric subunits of phosphofructokinase. As a result, band 3 not only rapidly activates the phosphofructokinase tetramer but also slowly inactivates the enzyme by preferentially binding its dissociated subunits.     

The interaction of calmodulin with erythrocyte membrane proteins

The method of sedimentation equilibrium in an air-driven ultracentrifuge (Airfuge) has been employed to investigate the interaction of 125I-calmodulin with the cytoskeletal components of the human red cell membrane. The results indicate significant calcium-dependent calmodulin binding activity in the low and high ionic strength extracts of the human erythrocyte membrane. The interaction of 125I-calmodulin with the low ionic strength extract proteins is analysed quantitatively. Further purification of the high ionic strength extract comprising mainly band 2.1 and band 4.1 results in the elution of calmodulin binding activity in a purified fraction of band 4.1.     

The interaction of short-chain aralkyl alcohols and amines with the erythrocyte membrane.

Erythrocytes in isotonic saline are hemolyzed by benzyl alcohol and by 2-phenylethanol, but not by the corresponding amines nor by the ring-or side-chain-hydroxylated analogs. All these compounds could however interact with the erythrocyte membrane since: a) they facilitated the hemolytic effect of benzyl alcohol and/or of phenylelytic effect of benzyl alcohol and/or of phenylethanol; b) they exerted a protective effect against controlled hypotonic hemolysis.     

Chain length-dependent interaction of free fatty acids with the erythrocyte membrane

Free fatty acids protect erythrocytes against hypotonic haemolysis in a certain low concentration range and become haemolytic at higher concentrations. The chain length dependence of this biphasic behaviour was investigated using human erythrocytes. The results can be summarized as follows: (i) A critical minimum chain length is required for both effects. Octanoic acid (C8) and fatty acids with a shorter chain length do not have any effect on the osmotic resistance of erythrocytes. (ii) Decanoic acid (C10) decreases the extent of hypo-osmotic haemolysis and does not become haemolytic at higher concentrations. (iii) Dodecanoic acid (C12) represents the minimum chain length for the typical concentration-dependent biphasic behaviour with protection against hypo-osmotic haemolysis at a certain low concentration range and subsequent haemolysis at higher concentrations. (iv) Tetradecanoic acid (C14) exhibits two concentration ranges of protection against hypo-osmotic haemolysis, each followed by haemolytic concentrations. (v) The observed effects are not correlated with the critical micellar concentrations of the investigated fatty acids.     

Platelet‐activating factor interaction with the human erythrocyte membrane

Platelet‐activating factor (PAF) is a soluble signal messenger present in blood at nanomolar concentration. PAF has a wide spectrum of biological activities and is produced by and effective in different cell types. Owing to its important physiological role, we wanted to characterize membrane intercalation and interaction of PAF‐16 (1‐O‐hexadecyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine) by studying its capacity to induce during short‐term incubations at high concentrations cell shape alterations, phosphatidylserine exposure, and hemolysis in human erythrocytes. Our results showed that PAF‐16 at micromolar concentrations rapidly (≤1 min) induces stable but wash‐sensitive echinocytosis and hemolysis, but no substantial phosphatidylserine exposure. In conclusion, our study characterizes PAF‐16 as a highly membrane partitioning non‐permeable molecule accumulating in the outer membrane leaflet. These membrane interacting properties of PAF should, also at physiological concentrations, be important part of its nature as a membrane affector molecule. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:345–348, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20297     

Direct interaction of mepacrine with erythrocyte and platelet membrane phospholipid

Mepacrine has been used as an inhibitor of the activation of endogenous phospholipases in many systems. These endogenous phospholipases are important in the modification of the lipid environment of membrane proteins and in the release of locally active oxygenated arachidonic acid metabolites. In both human platelets and erythrocytes, mepacrine blocks the release of fatty acid from phospholipid by endogenous phospholipases. However, mepacrine also interacts directly with membrane phospholipids, primarily phosphatidylethanolamine, to form less polar derivatives. This interaction occurs rapidly and is maximal at concentrations of mepacrine greater than 0.2 mM. Such drug-phospholipid interaction may perturb membrane architecture and function and be responsible for the inhibitory effects of mepacrine on cellular responses observed in many systems. Since the alteration in membrane phospholipid composition occurs under the same conditions as phospholipase inhibition, it is not possible to be certain that the inhibition of cellular responses by mepacrine is due to inhibition of phospholipases rather than to direct perturbation of the membrane. It is also possible that inhibition of phospholipase action by mepacrine is in part a consequence of the change in phospholipid composition. These results indicate that caution should be exercised in the interpretation of results obtained using mepacrine and that the usefulness of this compound for the investigation of the biological importance of phospholipase activation is limited.     

Human erythrocyte calmodulin. Further chemical characterization and the site of its interaction with the membrane.

Human erythrocyte and bovine brain calmodulins were indistinguishable by tryptic peptide mapping, indicating that the primary sequence of the two proteins is either very similar or identical. Calcium binding determinations of human erythrocyte calmodulin, by equilibrium dialysis and fluorescence titration, were in close agreement with previous studies on other calmodulins. The calcium-activated adenosine triphosphatase which is stimulated by calmodulin was shown to be firmly associated with smooth erythrocyte plasma membranes devoid of spectrin and actin. Kinetic titration demonstrated that there are 4500 calmodulin binding sites per erythrocyte and that the turnover number of this calcium-activated adenosine triphosphatase is 3000 mumol of Pi . (mumol of site)-1 . min-1 which is similar to the turnover numbers of other transport adenosine triphosphatases. Furthermore, calmodulin stimulates calcium-activated adenosine triphosphatase by a simple enzyme-ligand association.     

Temperature-dependent specificity of cis-trans isomeric fatty acid interaction with the erythrocyte membrane

Stabilization of red cells against hypotonic haemolysis by cis-trans isomeric free C18 fatty acids occurs with pronounced specificity which is strongly temperature-dependent, but in a distinctly different manner for the two configurational isomers. Oleic acid (cis-18:1) stabilizes very efficiently at 0 degrees C, even at the highest concentrations. Elaidic acid (trans-18:1) causes neither stabilization nor haemolysis at this temperature. At room temperature (23 degrees C), elaidic acid acquires the ability to protect, without turning haemolytic at high concentrations. At 37 degrees C elaidic acid also becomes haemolytic. The protecting effect of oleic acid at 0 degrees C is the result of a rapid reaction. The characteristic, temperature-dependent specificity of cis-trans isomeric C18 fatty acid interaction with the red cell membrane appears to be a general phenomenon, since it was observed alike with erythrocytes of different species.     

Critical temperatures for the interaction of free fatty acids with the erythrocyte membrane

Non-esterified long-chain fatty acids reduce the extent of hypotonic hemolysis at a certain low concentration range but cause hemolysis at higher concentrations. This biphasic behavior was investigated at different temperatures (0-37 degrees C) for lauric (12:0), myristic (14:0), palmitoleic (16:1), oleic (cis-18:1) and elaidic (trans-18:1) acids. The results are summarized as follows: (A) the fatty acids examined exhibit a high degree of specificity in their thermotropic behavior; (B) oleic acid protects against hypotonic hemolysis even at the highest concentrations, up to 15 degrees C, when it becomes hemolytic, but only in a limited concentration range; (C) elaidic acid does not affect the osmotic stability of erythrocytes up to 20 degrees C, when it starts protecting: above 30 degrees C, it becomes hemolytic at the highest concentrations; (D) palmitoleic acid is an excellent protecting agent at all temperatures in a certain concentration range, becoming hemolytic at higher concentrations; (E) lauric acid protects up to 30 degrees C and becomes hemolytic only above this temperature; (F) myristic acid exhibits an extremely unusual behavior at 30 and 37 degrees C by having alternating concentration ranges of protecting and hemolytic effects; (G) there is a common critical temperature for hemolysis at 30 degrees C for saturated and trans-unsaturated fatty acids; (H) the initial slope of Arrhenius plots of percent hemolysis at the concentration of maximum protection is negative for cis-unsaturated fatty acids and positive for saturated and trans-unsaturated fatty acids.     

E.s.r. radiation studies of erythrocyte membrane haemoglobin interaction.

The dependence of the yield of free radicals in gamma-irradiated, freeze-dried erythrocyte membranes on their haemoglobin content was studied. A non-monotonous relationship was found--different from that observed in mixtures of freeze-dried membranes and haemoglobin, which suggests the existence of radiation-energy transfer between the membranes and bound haemoglobin.     

Effect of detergents on antibody-antigen interaction.

The effect of the different detergent mixtures on immunodiffusion and immunoprecipitation was studied. The anionic detergent sodium dodecyl sulfate at concentrations above 0.2% ($\text{w}\text{v}$) inhibits the reaction between antigen and antibody by more than 90%. Nonionic detergents at a concentration of 1% ($\text{w}\text{v}$) have little or no detectable effect. In contrast, when we used mixtures of various concentrations of ionic and nonionic detergents the inhibitory effect of the ionic detergent decreased.     

The interaction of Triton X-100 with purple membranes. Detergent binding, spectral changes and membrane solubilization

The interaction of the non-ionic surfactant Triton X-100 with Halobacterium purple membranes has been examined at sublytic and lytic surfactant concentrations. These membranes present a number of important peculiarities in their behaviour towards the surfactant. Although solubilization is a very slow process, with a half-time of the order of hours, detergent binding appears to occur at the same fast rate as that found in other membranes. Lipids are solubilized more easily than proteins, so that hardly any protein is solubilized at surfactant concentrations at which about 75% of the lipid is in the form of detergent-mixed micelles; once started, protein solubilization takes place within a narrow range of surfactant concentrations. Retinal provides a built-in probe to monitor detergent-induced conformational changes by spectroscopy in the visible range. No spectral variation is detected at the prelytic stage, i.e. when detergent is incorporated into the membrane in monomeric form. Membrane disruption is accompanied by a blue shift in the absorption maximum, retinal isomerization (from all-trans to 13-cis), and a decrease in specific absorbance (bleaching). Increasing detergent concentrations after solubilization is completed do not produce further shifts in the spectral maximum, but the specific absorbance is progressively decreased. It is shown that Triton X-100 has a complex effect on the retinal chromophore, modifying its configuration and microenvironment (changes in maximum wavelength) and promoting hydrolysis of the retinal-bacteriorhopsin Schiff's base (bleaching).