5-doxylstearate and Hemolysis

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to monitor membrane dynamic changes in erythrocytes subjected to oxidative stress with hydrogen peroxide (H(2)O(2)). The lipid spin label, 5-doxyl stearic acid, responded to dramatic reductions in membrane fluidity, which was correlated with increases in the protein content of the membrane. Membrane rigidity, associated with the binding of hemoglobin (Hb) to the erythrocyte membrane, was also indicated by a spin-labeled maleimide, 5-MSL, covalently bound to the sulfhydryl groups of membrane proteins. At 2% hematocrit, these alterations in membrane occurred at very low concentrations of H(2)O(2) (50 µM) after only 5 min of incubation at 37°C in azide phosphate buffer, pH 7.4. Lipid peroxidation, suggested by oxidative hemolysis and malondialdehyde formation, started at 300 µM H(2)O(2) (for incubation of 3 h), which is a concentration about six times higher than those detected with the probes. Ascorbic acid and α-tocopherol protected the membrane against lipoperoxidation, but did not prevent the binding of proteins to the erythrocyte membrane. Moreover, the antioxidant (+)-catechin, which also failed to prevent the cross-linking of cytoskeletal proteins with Hb, was very effective in protecting erythrocyte ghosts from lipid peroxidation induced by the Fenton reaction. This study also showed that EPR spectroscopy can be useful to assess the molecular dynamics of red blood cell membranes in both the lipid and protein domains and examine oxidation processes in a system that is so vulnerable to oxidation.

Topics: alpha-Tocopherol; Antioxidants; Ascorbic Acid; Catechin; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Erythrocyte Membrane; Hemoglobins; Hemolysis; Humans; Hydrogen Peroxide; Hydrogen-Ion Concentration; Lipid Peroxidation; Membrane Fluidity; Membrane Proteins; Oxidative Stress; Thiobarbituric Acid Reactive Substances

Alyteserin-1c (GLKEIFKAGLGSLVKGIAAHVAS.NH(2)), first isolated from skin secretions of the midwife toad Alytes obstetricans, shows selective growth-inhibitory activity against Gram-negative bacteria. The structures of alyteserin-1c and its more potent and less haemolytic analogue [E4K]alyteserin-1c were investigated in various solution and membrane mimicking environments by proton NMR spectroscopy and molecular modelling. In aqueous solution, the peptide displays a lack of secondary structure but, in a 2,2,2-trifluoroethanol (TFE-d(3))-H(2)O solvent mixture, the structure is characterised by an extended alpha helix between residues Leu(2) and Val(21). Solution structural studies in the membrane mimicking environments, sodium dodecyl sulphate (SDS), dodecylphosphocholine (DPC), and 1,2-dihexanoyl-sn-glycero-3-phosphatidylcholine (DHPC) micelles, indicate that these peptides display an alpha helical structure between residues Lys(3) and Val(21). Positional studies of the peptides in SDS, DPC and DHPC media show that the N-terminal and central residues lie inside the micelle while C-terminal residues beyond Ala(19) do not interact with the micelles.

Topics: Anti-Infective Agents; Antimicrobial Cationic Peptides; Bacteria; Chlorides; Cyclic N-Oxides; Hemolysis; Humans; Lysine; Manganese Compounds; Membrane Lipids; Membranes, Artificial; Micelles; Microbial Sensitivity Tests; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Phospholipid Ethers; Phosphorylcholine; Protein Conformation; Sodium Dodecyl Sulfate; Solvents; Spin Labels; Structure-Activity Relationship; Trifluoroethanol; Valine; Water

We examined the effects of the double-chained cationic surfactants dimethyidialkylammoniums (CH3)2N+ (CnH(2n+1))2 on the permeation of benzoic acid through excised guinea pig dorsal skin. Among five dimethyldialkylammoniums tested (n=10-18), dimethyldidecylammonium (n=10) had dose-dependent enhancement effects at concentrations of more than 20 microM. Compared with the marked enhancement effects of dimethyldialkylammoniums with relatively shorter alkyl chains, those of long-chain dimethyldialkylammoniums (n=16, 18) were much less. We compared the enhancement effects of these cationic surfactants on skin permeation with their hemolytic effects on guinea pig erythrocytes. Their enhancement effects corresponded to their hemolytic activity. The findings suggest that dimethyldialkylammoniums with relatively shorter alkyl chains, which form either vesicles with looser molecular packing or micelles and appear to be present as surfactant monomers in higher ratios than those with longer alkyl chains, favor the interaction with skin. Their enhancement mechanism is possibly similar to that of single-chained cationic surfactants.

Topics: Algorithms; Animals; Benzoic Acid; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Erythrocytes; Guinea Pigs; Hemolysis; In Vitro Techniques; Quaternary Ammonium Compounds; Skin Absorption; Solubility; Spin Labels; Surface-Active Agents; Ultrasonics

The nature of the membranolytic interaction between monosodium urate monohydrate (MSUM) crystals and phospholipid membranes was studied using electron spin resonance. Two spin probe molecules were incorporated into intact human erythrocytes and incubated with MSUM crystals. The apparent increased fluidity of 5-doxyl stearic acid incorporated erythrocytes after a 2 h incubation with MSUM was probably due to an electrostatically induced redistribution of probe from the outer more rigid layer to the fluid inner leaflet via a flip-flop mechanism. It was suggested that the MSUM induced redistribution of cationic amphiphilic probe population in the whole erythrocyte was also due to an electrostatic interaction between negatively charged MSUM crystals and positively charged probe. Possible mechanisms of MSUM induced membranolysis are discussed.

Topics: Crystallization; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Erythrocyte Membrane; Erythrocytes; Hemolysis; Humans; Microscopy, Electron, Scanning; Spin Labels; Uric Acid