Small-angle X-ray scattering and electron paramagnetic resonance study of the interaction of bovine serum albumin with ionic surfactants

Small-angle X-ray scattering (SAXS) and electron paramagnetic resonance (EPR) techniques have been used to monitor the interaction of bovine serum albumin (BSA) with ionic surfactants such as anionic sodium dodecyl sulfate (SDS), zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonium-1-propane sulfonate (HPS), and cationic cethyltrimethylammonium chloride (CTAC) at pH 7.0. The SAXS results have shown that in the presence of 5 mM SDS and HPS the radius of gyration (Rg) almost does not change as compared to the BSA free-surfactant solution; its value is ca. 30 Angstroms. In the presence of 5 mM CTAC the SAXS data indicate the presence of a particle with a Rg of at least 63 Angstroms, suggesting that in this case, a kind of protein aggregation takes place. In the presence of SDS and HPS surfactants at concentrations above 10 mM, a characteristic broad peak in the region of 0.12-0.18 Angstroms(-1) indicates the presence of micelle-like aggregates in solution. The SAXS curves are consistent with the "pearl necklace" model, where micelle-like aggregates are randomly distributed around the polypeptide chain. EPR results using 5-DSA and 16-DSA spin labels show that in the presence of BSA the EPR spectra are composed of two label populations, one contacting the protein and a second one due to label localization in the micelles. Evidence is also obtained for a competition of the surfactants with the spin labels for the high-affinity binding sites of the stearic acid spin labels as monitored by changes in the fractions of the two label populations as the surfactant concentration is increased. The effect of SDS seems to be stronger in the sense that increased SDS concentration leads to a complete transfer of spin labels from close protein contact sites to micelles, while for HPS, a significant immobilization of probe apparently remains even at higher surfactant concentrations. These two techniques are quite useful since SAXS monitors the overall properties of the scattering particle, while EPR gives information on the dynamics inside this particle and associated with label localization and motion.     

Magnetic properties of metastable Gd-Cr alloys

We report on the magnetization, magnetocaloric effect, magnetic ordering temperatures, saturation magnetic moments and anisotropy of sputter-deposited Gd_xCr_1−x alloys with Gd atomic concentrations, x, ranging from 0.13 to 0.52. The complex magnetic nature of the Gd-Cr films was revealed from the M×H isotherms, which do not show saturation even at an applied field of 70 kOe and a temperature of 2 K and do not exhibit a linear behavior at higher temperatures. For some of the samples, the isotherms were used to determine the isothermal entropy variation as a function of temperature, for a change of 50 kOe in the applied magnetic field. The saturation magnetic moment varies with x and follows the dilution law, implying that the Cr atoms do not contribute to the total moment of the Gd-Cr alloys. Both static magnetization and dynamic susceptibility measurements reveal the existence of a magnetic glassy behavior in the alloys, which occurs below a freezing temperature. The existence of anisotropy at low temperatures for all samples was revealed by their M×H hysteresis loops from which the in-plane coercive fields, H_c, were determined. A monotonical increase in H_c with increasing Gd concentration was observed.     

Chlorpromazine and sodium dodecyl sulfate mixed micelles investigated by small angle X-ray scattering

Small-angle X-ray scattering (SAXS) studies are reported on the interaction of chlorpromazine (CPZ) with micelles of anionic surfactant sodium dodecyl sulfate (SDS). Isotropic solutions of SDS (40 and 100 mM) at pH 4.0, 7.0, and 9.0 in the absence and presence of CPZ (2-25 mM) were investigated at the National Laboratory of Synchrotron Light (LNLS, Campinas, Brazil). The data were analyzed through the modeling of the micellar form factor and interference function. The results evidence a micellar shape transformation from prolate ellipsoid to cylinder accompanied by micellar growth and surface charge screening as the molar ratio CPZ : SDS increases in the complex. Small ellipsoids with axial ratio nu=1.5+/-0.1 at 40 mM SDS grow and reassemble into cylinder-like aggregates upon 5 mM drug incorporation (1 CPZ : 8 SDS monomers) with a decrease of the micelle surface charge. At 10 mM CPZ : 40 mM SDS cylindrical micelles are totally screened with an axial ratio nu approximately 2.5. The data also indicate the presence of small prolate ellipsoids (nu=1.7+/-0.1) in solutions of 100 mM SDS (no drug) and micellar growth (nu approximately 2.0 and 4.0) when 10 and 25 mM CPZ are added to the system. In the latter case, the aggregate is also better represented by a cylinder-like form. Therefore, our results demonstrate that the axial ratio and shape evolution of the surfactant : phenothiazine complex are both SDS concentration and drug : SDS molar ratio dependent. The drug location close to the SDS polar headgroup region without disrupting in a significant way both the paraffinic hydrophobic core and the polar shell thickness is inferred. SAXS data made it possible to obtain the shapes and dimensions of CPZ/SDS aggregates.     

Spectroscopic studies on the interaction of bovine (BSA) and human (HSA) serum albumins with ionic surfactants

Bovine (BSA) and human (HSA) serum albumins are frequently used in biophysical and biochemical studies since they have a similar folding, a well known primary structure, and they have been associated with the binding of many different categories of small molecules. One important difference of BSA and HSA is the fact that bovine albumin has two tryptophan residues while human albumin has a unique tryptophan. In this work results are presented for the interaction of BSA and HSA with several ionic surfactants, namely, anionic sodium dodecyl sulfate (SDS), cationic cethyltrimethylammonium chloride (CTAC) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate (HPS), as monitored by fluorescence spectroscopy of intrinsic tryptophans and circular dichroism spectroscopy. On the interaction of all three surfactants with BSA, at low concentrations, a quenching of fluorescence takes place and Stern-Volmer analysis allowed to estimate their 'effective' association constants to the protein: for SDS, CTAC and HPS at pH 7.0 these constants are, respectively, (1.4+/-0.1) x 10(5) M(-1), (8.9+/-0.1) x 10(3) M(-1) and (1.4+/-0.1) x 10(4) M(-1). A blue shift of maximum emission is observed from 345 to 330 nm upon surfactant binding. Analysis of fluorescence emission spectra allowed to separate three species in solution which were associated to native protein, a surfactant protein complex and partially denatured protein. The binding at low surfactant concentrations follows a Hill plot model displaying positive cooperativity and a number of surfactant binding sites very close to the number of cationic or anionic residues present in the protein. Circular dichroism data corroborated the partial loss of secondary structure upon surfactant addition showing the high stability of serum albumin. The interaction of the surfactants with HSA showed an enhancement of fluorescence at low concentrations, opposite to the effect on BSA, consistent with the existence of a unique buried tryptophan residue in this protein with considerable static quenching in the native state. The effects of surfactants at low concentrations were very similar to those of myristic acid suggesting a non specific binding through hydrophobic interaction modulated by eletrostatic interactions. The changes in the vicinity of the tryptophan residues are discussed based on the recently published crystallographic structure of HSA myristate complex (S. Curry et al., Nat. Struct. Biol. 5 (1998) 827).