Influence of the temperature in the adsorption of sodium dodecyl sulfate on phosphatidylcholine liposomes

The influence of the temperature on the adsorption of monomeric and micellar solutions of the anionic surfactant sodium dodecyl sulfate (SDS) on phosphatidylcholine (PC) liposomes was investigated using the fluorescent probe 2-(p-toluidinyl)-naphthalene-6-sodium sulfonate (TNS). The number of adsorbed molecules was quantified by measuring changes in the electrostatic potential (Psi(o)) of the liposomes/probe during an incubation with SDS at varying temperatures. At low surfactant concentrations (from 0.05 to 0.25 mM), the increase in temperature reduced the number of surfactant molecules incorporated per vesicle regardless of the incubation time, whereas at high surfactant concentrations (from 0.50 to 1.0 mM) the incubation time has an opposite effect on this process. Thus, after 10s, the surfactant adsorption decreased with temperature, yet it increased progressively with time. The adsorption was linear with temperature below critical micellar concentration (CMC) of SDS and this linear tendency did not change above CMC. This suggests an adsorption of SDS monomers regardless of the surfactant concentration.     

Subunit structure of the major human erythrocytes glycoprotein: depolymerization by heating ghosts with sodium dodecyl sulfate

Heating human erythrocyte ghosts with sodium dodecyl sulfate (SDS) at 100° was found to cause depolymerization of the major membrane glycoprotein. The molecular weight of the heat-induced product was found to be about half that of the precursor and to have an identical surface charge density in SDS. These findings were obtained by analysis of Ferguson plots derived from SDS- polyacrylamide gel electrophoresis in which the retardation coefficients and free mobilities of the two glycoprotein forms were compared. Based on these findings, we propose that previous conflicts regarding the molecular weight of this glycoprotein can be resolved by reference to the isolation and/or solubilization conditions.     

A precise spectrophotometric method for measuring sodium dodecyl sulfate concentration

Sodium dodecyl sulfate (SDS) is used to denature and solubilize proteins, especially membrane and other hydrophobic proteins. A quantitative method to determine the concentration of SDS using the dye Stains-All is known. However, this method lacks the accuracy and reproducibility necessary for use with protein solutions where SDS concentration is a critical factor, so we modified this method after examining multiple parameters (solvent, pH, buffers, and light exposure). The improved method is simple to implement, robust, accurate, and (most important) precise.     

Effects of temperature and bovine serum albumin on lysis of erythrocytes induced by dilauroylglycerophosphocholine and didecanoylglycerophosphocholine.

The effects of the incubation temperature and bovine serum albumin on hemolysis induced by short-chain phosphatidylcholine were examined. The rate of hemolysis of human, monkey, rabbit, and rat erythrocytes by dilauroylglycerophosphocholine showed biphasic temperature-dependence: hemolysis was rapid at 5-10 degrees C and above 40 degrees C, but slow at around 25 degrees C. In contrast, the rate of lysis of cow, calf, sheep, pig, cat, and dog erythrocytes did not show biphasic temperature-dependence, but increased progressively with increase in the incubation temperature. Bovine serum albumin increased the hemolysis of human erythrocytes induced by dilauroylglycerophosphocholine or didecanoylglycerophosphocholine: it shortened the lag time of lysis and reduced the amount of phosphatidylcholine required for lysis. A shift-down of the incubation temperature from 40 to below 10 degrees C also shortened the lag time of lysis of human erythrocytes induced by dilauroylglycerophosphocholine and reduced the amount of phosphatidylcholine required for lysis.     

Inhibition of amyloid aggregation of bovine serum albumin by sodium dodecyl sulfate at submicellar concentrations

Sodium dodecyl sulfate (SDS), as an anionic surfactant, can induce protein conformational changes. Recent investigations demonstrated different effects of SDS on protein amyloid aggregation. In the present study, the effect of SDS on amyloid aggregation of bovine serum albumin (BSA) was evaluated. BSA transformed to β-sheet-rich amyloid aggregates upon incubation at pH 7.4 and 65°C, as demonstrated by thioflavin T fluorescence, circular dichroism, and transmission electron microscopy. SDS at submicellar concentrations inhibited BSA amyloid aggregation with IC₅₀ of 47.5 μM. The inhibitory effects of structural analogs of SDS on amyloid aggregation of BSA were determined to explore the structure–activity relationship, with results suggesting that both anionic and alkyl moieties of SDS were critical, and that an alkyl moiety with chain length ≥10 carbon atoms was essential to amyloid inhibition. We attributed the inhibitory effect of SDS on BSA amyloid aggregation to interactions between the detergent molecule and the fatty acid binding sites on BSA. The bound SDS stabilized BSA, thereby inhibiting protein transformation to amyloid aggregates. This study reports for the first time that the inhibitory effect of SDS on albumin fibrillation is closely related to its alkyl structure. Moreover, the specific binding of SDS to albumin is the main driving force in amyloid inhibition. This study not only provides fresh insight into the role of SDS in amyloid aggregation of serum albumin, but also suggests rational design of novel antiamyloidogenic reagents based on specific-binding ligands.     

The effect of temperature on lysis of human erythrocytes by palmitic acid]

An analysis of kinetic curves of erythrocyte hemolysis induced by palmitic acid has shown the existence of some stages of this process. The activation energy of hemolysis, as determined by the temperature dependence of the hemolysis rate constant, was 210 +/- 30 kJ/mol. It was shown by the method of stepwise thermoinactivation of erythrocytes proteins that at temperature of 49 degrees C which corresponded to the framework protein spectrin denaturation temperature, the erythrocyte membrane stability sharply decreased. On the contrary, changes of the cell shape induced by the hyperosmotic medium (0.5 M sucrose) inhibited the palmitic acid-induced erythrocytes hemolysis.     

Denaturation of MM-creatine kinase by sodium dodecyl sulfate

The denaturation of dimeric cytoplasmic MM-creatine kinase by sodium dodecyl sulfate (SDS) has been investigated using activity measurements, far-ultraviolet circular dichroism, SEC-HPLC, electric birefringence, intrinsic probes (cysteine and tryptophan residues), and an extrinsic fluorescent probe (ANS). Our results show that inactivation is the first detectable event; the inactivation curve midpoint is located around 0.9 mM SDS. The second event is dissociation and it occurs in parallel to tertiary and secondary perturbations, as demonstrated by the coincidence (near 1.3 mM) of the midpoints of the transition curves monitoring dissociation and structural changes. At high total SDS concentration (concentration higher than 2.5 mM), the monomer had bound 170 mol of SDS per mol of protein. In these conditions, electric birefringence experiments suggest that the SDS-CK complex may be described as a prolate ellipsoid with an axial ratio of 1.27 (14 nm×11 nm). These results are compatible with recent models of SDS-protein complexes: the protein decorated micelle structure or the necklace structure.     

Inactivation of prions by acidic sodium dodecyl sulfate

Prompted by the discovery that prions become protease-sensitive after exposure to branched polyamine dendrimers in acetic acid (AcOH) (S. Supattapone, H. Wille, L. Uyechi, J. Safar, P. Tremblay, F. C. Szoka, F. E. Cohen, S. B. Prusiner, and M. R. Scott, J. Virol. 75:3453-3461, 2001), we investigated the inactivation of prions by sodium dodecyl sulfate (SDS) in weak acid. As judged by sensitivity to proteolytic digestion, the disease-causing prion protein (PrPSc) was denatured at room temperature by SDS at pH values of < or =4.5 or > or =10. Exposure of Sc237 prions in Syrian hamster brain homogenates to 1% SDS and 0.5% AcOH at room temperature resulted in a reduction of prion titer by a factor of ca. 10(7); however, all of the bioassay hamsters eventually developed prion disease. When various concentrations of SDS and AcOH were tested, the duration and temperature of exposure acted synergistically to inactivate both hamster Sc237 prions and human sporadic Creutzfeldt-Jakob disease (sCJD) prions. The inactivation of prions in brain homogenates and those bound to stainless steel wires was evaluated by using bioassays in transgenic mice. sCJD prions were more than 100,000 times more resistant to inactivation than Sc237 prions, demonstrating that inactivation procedures validated on rodent prions cannot be extrapolated to inactivation of human prions. Some procedures that significantly reduced prion titers in brain homogenates had a limited effect on prions bound to the surface of stainless steel wires. Using acidic SDS combined with autoclaving for 15 min, human sCJD prions bound to stainless steel wires were eliminated. Our findings form the basis for a noncorrosive system that is suitable for inactivating prions on surgical instruments, as well as on other medical and dental equipment.     

Parameters of interaction of sodium dodecyl sulfate with human hemoglobin

It was shown that sodium dodecyl sulfate at concentrations not exceeding the critical micelle concentrations (0-1.9 mM) induced the conversion of oxy- and methemoglobin but not deoxyhemoglobin to hemichrome. The concentration dependences of hemichrome formation were represented as Hill plots, and the parameters of detergent binding were estimated. OxyHb in 20 mM potassium-phosphate buffer, pH 6.8, has two groups of binding sites: the first group is characterized by the Hill constant n1 = 2 and the concentration of half saturation [SDS]50 = 0.8 mM, and the second group is characterized by the Hill constant n2 = 8 and [SDS]50 = 0.9 mM. In the case of metHb one group of binding sites with the Hill constant n = 2 and half saturation concentration [SDS]50 = 0.2 mM was observed. An increase in environmental pH to 7.9 decreased the affinity of Hb for SDS. It is suggested that primary binding sites for SDS in oxyHb coincide with the anion-binding center of the Hb molecule. The interaction of the detergent with these binding sites induced a structural transition of the hemoprotein molecule. As a result of this transition, secondary binding sites were exposed. In a model system (hemin--imidazole in ethanol solution), the enthalpy of the transition of hemin from a high-spin to a low-spin state was estimated to be 47 +/- 7 kJ/mol.     

Interactions between bovine plasma albumin and sodium dodecyl sulfate studied by means of 13C-NMR spectra.

The interactions between the protein, bovine plasma albumin, and surfactant, sodium dodecyl sulfate, have been studied by ¹³C-nmr spectroscopy at pH 5.4–6.8 in D₂O solution. The ¹³C chemical shifts and the ¹³C spin-lattice relaxation time of the individual carbons of the surfactant were measured as a function of the molar ratio of the surfactant to albumin in order to analyze the surfactant-protein interaction and the molecular motion of the surfactant. It was found that in the region of initial binding of the surfactant to the high-affinity sites on the protein, both the surfactant head group and alkyl chain interact with the protein. With an excess of high-affinity sites at the beginning of the reaction, surfactant molecules are in a micellelike environment in which the surfactant's alkyl chains are associated with nonpolar groups of the protein. Even after the denaturation by many surfactant bindings, much of the secondary and higher structure seems to remain intact.     

Influence of heat and sodium dodecyl sulfate on the endopeptidase I from Bacillus sphaericus 9602

Endopeptidase I from Bacillus sphaericus is a stable enzyme which retains its activity at 37 degrees C in the presence of sodium dodecyl sulfate. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed two forms of the enzyme: an active, fast-running form, for the enzyme preheated at 37 degrees C and a denatured, slow-running form, for the enzyme preheated at 100 degrees C. Such behavior is similar to that of the "heat-modifiable" outer membrane proteins from gram-negative bacteria. In the absence of sodium dodecyl sulfate, endopeptidase I aggregated in an enzymatically active dimer, with an apparent molecular weight of 90,000 daltons, which could be the native form of the enzyme.     

Conformation and aggregation of melittin: effect of pH and concentration of sodium dodecyl sulfate

The conformation of melittin, a surface-active polypeptide, in solution was studied by CD spectra between 190 and 240 nm. The molecule was essentially unordered (possibly with a trace of helix) in water without salt at neutral pH. Upon deprotonation of four of the six cationic groups at pH 12 the polypeptide became partially helical (about 35%). The addition of NaDodSO₄ to an aqueous melittin solution first caused the solution to become turbid but it became clear again in excess surfactant solution. The conformational changes depended on the molar NaDodSO₄/melittin ratio, R. With R from 2.34 to 23.4, the melittin solution was turbid and the polypeptide conformation was probably a mixture of α-helix and β-sheets. This was supported by the ir spectrum of the turbid solution, which indicated the presence of both conformations. With R = 46.8 or 468 (1 or 10 mM NaDodSO₄) the polypeptide conformation was characteristic of an α-helix, about 70–80% of the molecule, regardless of whether the surfactant was above or below its critical micelle concentration. This compared well with the x-ray results of 92% helix in crystals. The lower helicity of melittin in NaDodSO₄ solution might be attributed to the end effects that destabilize the first and last turn of an helix at its N- and C-terminus, respectively.