The binding of sodium dodecyl sulphate to bovine serum albumin at high binding ratios

The extent of binding of sodium dodecyl sulphate to bovine serum albumin at high binding ratios was investigated by gel filtration. The weight ratio of bound sodium dodecyl sulphate to bovine serum albumin increases with the NaCl concentration, and, except at low salt concentrations, with the concentration of sodium dodecyl sulphate. In the presence of 1.0g of sodium dodecyl sulphate/l, the binding ratio varied from 1.0 (at 0.04m-Na(+)) to 2.2 (at 0.44m-Na(+)). In the presence of 0.24m-Na(+), the binding ratio increased with sodium dodecyl sulphate concentration, from 0.9 (0.2g of sodium dodecyl sulphate/l) to 2.0 (5g of sodium dodecyl sulphate/l), at 26 degrees C, in a dilute sodium phosphate buffer. No significant dependence of the binding ratio upon temperature in the range 26-45 degrees C was observed. These results differ from those of Reynolds & Tanford (1970a) obtained by equilibrium dialysis.     

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.     

Low concentrations of sodium dodecyl sulfate induce the extension of beta 2-microglobulin-related amyloid fibrils at a neutral pH

In beta(2)-microglobulin-related (Abeta2M) amyloidosis, partial unfolding of beta(2)-microglobulin (beta2-m) is believed to be prerequisite to its assembly into Abeta2M amyloid fibrils in vivo. Although low pH or 2,2,2-trifluoroethanol at a low concentration has been reported to induce partial unfolding of beta2-m and subsequent amyloid fibril formation in vitro, factors that induce them under near physiological conditions have not been determined. Using fluorescence spectroscopy with thioflavin T, circular dichroism spectroscopy, and electron microscopy, we here show that at low concentrations, sodium dodecyl sulfate (SDS) converts natively folded beta2-m monomers into partially folded, alpha-helix-containing conformers. Surprisingly, this results in the extension of Abeta2M amyloid fibrils at neutral pH, which could be explained basically by a first-order kinetic model. At low concentrations, SDS also stabilized the fibrils at neutral pH. These SDS effects were concentration-dependent and maximal at approximately 0.5 mM, around the critical micelle concentration of SDS (0.67 mM). As the concentration of SDS was increased above 1 mM, the alpha-helix content of beta2-m rose to approximately 10%, while the beta-sheet content decreased to approximately 20%, a change paralleled by a complete cessation of fibril extension and the destabilization of the fibrils. Detergents of other classes had no significant effect on the extension of fibrils. These findings are consistent with the hypothesis that in vivo, specific factors (e.g., phospholipids) that affect the conformation and stability of beta2-m and amyloid fibrils will have significant effects on the kinetics of Abeta2M fibril formation.     

Thermal aggregation of bovine serum albumin at different pH: comparison with human serum albumin

We report here a study on thermal aggregation of BSA at two different pH values selected to be close to the isoelectric point (pI) of this protein. Our aim is to better understand the several steps and mechanisms accompanying the aggregation process. For this purpose we have performed kinetics of integrated intensity emission of intrinsic and extrinsic dyes, tryptophans and ANS respectively, kinetics of Rayleigh scattering and of turbidity. The results confirm the important role played by conformational changes in the tertiary structure, especially in the exposure of internal hydrophobic regions that promote intermolecular interactions. We also confirm that the absence of electrostatic repulsion favours the disordered non-specific interactions between molecules and consequently affects the aggregation rate. Finally, the comparison between BSA and another relative protein, HSA, allows us to clarify the role of different domains involved in the aggregation process. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

Secondary structural changes of large and small fragments of bovine serum albumin in thermal denaturation and in sodium dodecyl sulfate denaturation

The helicities in various fragments of bovine serum albumin (BSA) were examined in the thermal denaturation and in sodium docecyl sulfate (SDS) denaturation. The thermal denaturation was examined in a temperature range between 2 and 65°C. The helicity decreased with a rise of temperature and it recovered to some degree upon cooling temperature. A rather high reversibility was observed in the BSA fragments, which were located in the N-terminal of the parent protein and then contained the first large loop with no disulfide bridge. The high reversibility was available also for the helicity in the first large loop of the fragment, disulfide bridges of which were reduced. The fragments, which were smaller than one domain, became unstable in the SDS denaturation. The helicities of such fragments decreased in lower SDS concentrations compared with those of the intact BSA and the large fragments, which contained one or more domains. A resistance to the SDS denaturation appeared in the helices of every large loop even after the fragmentation. On the other hand, helicities of the fragments decreased to 20–25% upon the reduction of disulfide bridges. However, the helicities of these fragments increased to 35–40% in the SDS denaturation.     

Thermal aggregation of glycated bovine serum albumin

Aggregation and glycation processes in proteins have a particular interest in medicine fields and in food technology. Serum albumins are model proteins which are able to self-assembly in aggregates and also sensitive to a non-enzymatic glycation in cases of diabetes. In this work, we firstly reported a study on the glycation and oxidation effects on the structure of bovine serum albumin (BSA). The experimental approach is based on the study of conformational changes of BSA at secondary and tertiary structures by FTIR absorption and fluorescence spectroscopy, respectively. Secondly, we analysed the thermal aggregation process on BSA glycated with different glucose concentrations. Additional information on the aggregation kinetics are obtained by light scattering measurements. The results show that glycation process affects the native structure of BSA. Then, the partial unfolding of the tertiary structure which accompanies the aggregation process is similar both in native and glycated BSA. In particular, the formation of aggregates is progressively inhibited with growing concentration of glucose incubated with BSA. These results bring new insights on how aggregation process is affected by modification of BSA induced by glycation.     

Stepwise degradation of serum low denisty lipoprotein by sodium dodecyl sulfate.

The structure of human serum low density lipoprotein (LDL) was investigated by perturbing the LDL structure with sodium dodecyl sulfate (SDS). The change in LDL structure induced by the addition of SDS was monitored by sedimentation velocity measurements, ultraviolet difference spectroscopy, fluorescence spectroscopy and proteolytic digestion of apo-LDL with subtilisin BPN' [EC 3.4.21.14]. As the concentration of SDS was increased from 0.1 mg/ml to 3 mg/ml with LDL concentrations between 2.0 mg/ml and 4.4 mg/ml, the sedimentation coefficient of LDL changed in three distinct steps. It was found by chemical analyses that not more than 30% of the total lipid was lost from LDL in the second step, whereas the final step in the change of sedimentation coefficient corresponded to the complete removal of apo-LDL from the constituent lipids of LDL. The ultraviolet difference spectrum between the native and SDS-treated LDL and the quenching of LDL fluorescence underwent about 80% of the total change while the SDS concentration was only sufficient to cause the second of the three step changes in sedimentation coefficient. SDS-polyacrylamide gel electrophoresis of apo-LDL treated with subtilisin BPN' also showed that more than 70% of apo-LDL became susceptible to proteolysis under the same conditions. These results were interpreted as indicating that the solubilization of 20 to 30% of the lipids on the surface of LDL exposed nearly 80% or more of apo-LDL to the solvent. A small portion of apo-LDL was, however, still firmly anchored to the remaining lipid micelle as long as the concentration of SDS was less than that required to cause the final step of the change in sedimentation coefficient.     

Differential scanning calorimetric studies on bovine serum albumin: III. Effect of sodium dodecyl sulphate

Measurements of differential scanning calorimetry (d.s.c.) have been made on the complex bovine serum albumin (BSA)--sodium dodecyl sulphate (SDS) under various conditions. There are two peaks P1 and P2 in the d.s.c. curve for BSA at pH 7 and in the absence of NaCl, indicating the presence of the heat-induced transition of BSA. There are three peaks P1, P2 and P3 in the curve for the system with the molar mixing ratio SDS/BSA = 1. With the increase in the amount of SDS, the peak P3 grows at the expense of P1 and P2. There is only a single peak P3 in the curve for the systems SDS/BSA > 7, and no peak at SDS/BSA = 50 and 100. There is a single peak P12 in the curve for BSA at pH 7 and in the presence of 0.05 M NaCl, indicating that the heat-induced transition is suppressed. There are two peaks P12 and P3 for the systems SDS/BSA = 1-5; the area ratio of the peak P3 to P12 increases with the increase in the amount of SDS. There is only a single peak P3 when SDS/BSA > 7, and no peak at SDS/BSA = 50. It is concluded that the peak P3 is a product of SDS regardless of the presence or absence of NaCl. Values of thermal denaturation temperature (Td) and enthalpy (delta H) of thermal denaturation indicate that the complex AD12 (A = BSA, D = SDS) is in the most thermostabilized state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of the blue form of bacteriohodopsin by low concentrations of sodium dodecyl sulfate

The effects produced on bacteriorhodopsin by low concentrations of several detergents have been studied by absorption and fourth-derivative spectrophotometry. Sodium dodecyl sulfate induces the appearance of the blue form of bacteriorhodopsin (λ_max = 600 nm) at pH values up to 7.0 in a reversible manner. The apparent pK of the purple-to-blue transition raised with increasing concentration of SDS. Of the other detergents tested, only sodium dodecyl-N-sarcosinate showed a slight red-shift of the absorption band to 580 nm, whereas sodium taurocholate, Triton X-100 and cetyltrimethylammonium bromide did not favour the appearance of the blue form. The effect of SDS was found to be consistent with a localized conformational change that moves away the counter-ion of the protonated Schiff base.     

Low sodium dodecyl sulfate concentrations inhibit tobacco mosaic virus coat protein amorphous aggregation and change the protein stability

Effects of low SDS concentrations on amorphous aggregation of tobacco mosaic virus (TMV) coat protein (CP) at 52 degrees C and on the protein structure were studied. It was found that SDS completely inhibits the TMV CP (11.5 microM) unordered aggregation at the detergent/CP molar ratio of 15 : 1 (0.005% SDS). As judged by fluorescence spectroscopy, these SDS concentrations did not prevent heating-induced disordering of the large-distance part of the TMV CP subunit, including the so-called "hydrophobic girdle". At somewhat higher SDS/protein ratio (40 : 1) the detergent completely disrupted the TMV CP hydrophobic girdle structure even at room temperature. At the same time, these low SDS concentrations (15 : 1, 40 : 1) strongly stabilized the structure of the small-distance part of the TMV CP molecule (the four alpha-helix bundle) against thermal disordering as judged by the far-UV (200-250 nm) CD spectra. Possible mechanisms of TMV CP heating-induced unordered aggregation initiation are discussed.     

Thermal aggregation and ion-induced cold-gelation of bovine serum albumin

Protein cold-gelation has recently received particular attention for its relevance in bio and food technology. In this work, we report a study on bovine serum albumin cold-gelation induced by copper or zinc ions. Metal-induced cold-gelation of proteins requires two steps: during the first one, the heat treatment causes protein partial unfolding and aggregation; then, after cooling the solution to room temperature, gels are formed upon the addition of metal ions. The thermally induced behaviour has been mainly investigated through different techniques: Fourier transform infrared (FTIR) spectroscopy, circular dichroism, dynamic light scattering (DLS) and rheology. Data have shown that the aggregation process is mainly due to protein conformational changes—α-helices into β-aggregates—forming small aggregated structures with a mean diameter of about 20 nm a few minutes after heating. After metal ion addition, the viscoelastic properties of the gels have been investigated by rheological measurements. The behaviour of the elastic and viscous moduli as a function of time is discussed in terms of ion concentration and type. Our results show that: (1) the elastic behaviour depends on ion concentration and (2) at a given ion concentration, gels obtained in the presence of zinc exhibit an elastic value larger than that observed in the Cu²⁺ case. Data suggest that cold-gelation is the result of different mechanisms: the ion-mediated protein–protein interaction and the bridging effect due to the presence of divalent ions in solution.     

Secondary structure transitions and aggregation induced in dynorphin neuropeptides by the detergent sodium dodecyl sulfate

Dynorphins, endogeneous opioid neuropeptides, function as ligands to the opioid kappa receptors and also induce non-opioid effects in neurons, probably related to direct membrane interactions. We have characterized the structure transitions of dynorphins (big dynorphin, dynorphin A and dynorphin B) induced by the detergent sodium dodecyl sulfate (SDS). In SDS titrations monitored by circular dichroism, we observed secondary structure conversions of the peptides from random coil to α-helix with a highly aggregated intermediate. As determined by Fourier transform infrared spectroscopy, this intermediate exhibited β-sheet structure for dynorphin B and big dynorphin. In contrast, aggregated dynorphin A was α-helical without considerable β-sheet content. Hydrophobicity analysis indicates that the YGGFLRR motif present in all dynorphins is prone to be inserted in the membrane. Comparing big dynorphin with dynorphin A and dynorphin B, we suggest that the potent neurotoxicity of big dynorphin could be related to the combination of amino acid sequences and secondary structure propensities of dynorphin A and dynorphin B, which may generate a synergistic effect for big dynorphin membrane perturbing properties. The induced aggregated α-helix of dynorphin A is also correlated with membrane perturbations, whereas the β-sheet of dynorphin B does not correlate with membrane perturbations.     

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.     

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.     

Assay of Na,K-ATPase in plasma membrane preparations: Increasing the permeability of membrane vesicles using sodium dodecyl sulfate buffered with bovine serum albumin

Determination of maximal Na,K-ATPase activity in isolated plasma membranes is generally hampered by the vesicular nature of the preparation, limiting access of ATP and ions to one face or the other of the transmembrane protein. Detergents are often used to make the vesicles permeable to the substrates; however, the detergent/protein ratio is extremely critical for optimal activation. The use of bovine serum albumin as a detergent buffer is described. With this method the amount of membrane protein in the assay can be varied over a wide range, with full detergent activation. The method has been used for assay of Na,K-ATPase activity of membranes from dog kidney, rabbit brain, and electric organ of eel.