Serum albumin is the most abundant protein in the circulatory system. The ability of albumins to undergo a reversible conformational transition, observed with changes in pH, is conserved in distantly related species, suggesting for it a major physiological role possibly related to the transport of small molecules including drugs. We have followed changes of bovine serum albumin (BSA) in volume by densimetry and in adiabatic compressibility during its conformational transition from pH 7-2, using ultrasound measurements. In parallel, circular dichroism was measured. The volume and adiabatic compressibility decrease from pH 4 to 2. The change in ellipticity shows a decrease over the same pH range from 70% to 40% of its alpha-helix content. Sorbitol, at concentrations from 0 to 2 M, led to the progressive restoration of BSA volume and compressibility values, as well as a substantial recovery of its original alpha-helix content. This finding implies that the compressibility variation observed reflects the conformational changes during the transition. The mutual interactions of the mechanical properties and structural features of BSA reported here are important in biotechnology for research in material sciences and for the design and the development of new, tailor-made drug carriers.

The self-assembly of amphiphilic molecules into supramolecular aggregates involves a number of complex phenomena and forces. Recent developments of highly sensitive, densimetric and acoustic methods on small volume samples have provided novel sensitive probes to explore the physical properties of these complex fluids. We have investigated, by high precision densimetry and ultrasound velocimetry, reverse micelles of [sodium bis(2-ethylhexyl)sulfosuccinate] in oil (isooctane and decane), at increasing water concentration and at variable micellar volume fractions. The size of these spherical micelles has been determined by small angle x-ray scattering. Using these results, in the framework of the effective medium theory, we have developed a simple model of micellar compressibility, allowing the calculation of physical parameters (aggregation number, volume, and compressibility) of the surfactant monomolecular film as well as that of the micellar waters. In particular, we show that the central aqueous core designated as "free" water, located at a distance from the oil-water interacting interface, is twice as compressible as "bulk" water. One notable feature of this work is the influence of the nature of the oil on the above parameters.

The solubility, reactivity, and conformational dynamics of myelin basic protein (MBP) from bovine brain were studied in reverse micelles of sodium bis(2-ethylhexyl) sulfosuccinate (AOT)-isooctane and water. Such a membrane-mimetic system resembles the aqueous spaces of native myelin sheath in terms of physicochemical properties as reflected in the high affinity of MBP for interfacial bound water. This is marked by the unusual profile of the solubility curve of the protein in reverse micelles, which shows optimal solubility at a much lower molar ratio of water to surfactant ([ H2O]/[AOT] = w0) than that reported for other water-soluble proteins. The role of counterions and/or charged polar head groups in the solubilization process is revealed by comparison of the solubility of MBP in nonionic surfactant micellar solutions. Whereas MBP is unfolded in aqueous solutions, insertion into reverse micelles generates a more folded structure, characterized by the presence of 20% alpha-helix. This conformation is unaffected by variations in the water content of the system (in the 2.0-22.4 w0 range). The reactivity of epsilon-amino groups of lysine residues with aqueous solutions of o-phthalaldehyde demonstrates that segments of the peptide chain are accessible to water. Similar results were obtained with the sequence involved in heme binding. In contrast, the sole tryptophan residue, Trp-117, is shielded from the aqueous solvent, as indicated by lack of reaction with N-bromosuccinimide. The invariance of the wavelength maximum emission in the fluorescence spectra as a function of w0 is consistent with this result.(ABSTRACT TRUNCATED AT 250 WORDS)

A complete experimental format is given for the reconstitution of human hemoglobin from the separated heme-free a-and -globin chains (a', ft') and hemin, by two alternative routes. Based on their oxygen binding properties, the reaction of the ferri-forms with reducing agent, and the response of the oxygen binding curves to pH variation and to the addition of the a losteric effector 2,3-diphosphoglycerate, the molecules are native. One reconstitution route uses direct addition of hemin to the separated globin chains with production of the separated subunits, which can then be recombined and reduced. This procedure occasions losses by precipitation in the heme-addition step except at hig dilutions, and the yields are low. In the second pathway, either globin chain is mixed with the complementary untreated subunit to form the half-filled (with heme) intermediates, which combine stoichiometrically with hemin. No precipitation accompanies these reactions. For a-globin, the yield is about 50% because of incomplete combination with the heme-containing P chain. For -globin, the yield is better than 70%. It is suggested that experiments intended to test either globin chain should use the second route in preparation for structural or functional comparisons with native hemoglobin.In two previous papers (1, 2), we described the preparation and properties of isolated heme-free human globin chains (a", a°) and of two kinds of reassembly intermediates in which both kinds of chains are present but only half of the heme sites are filled (HFa and HFft molecules). The first paper demonstrated that the separated globin chains near neutral pH have different conformations, a" being substantially more disordered than a", in contrast to the separated heme-containing subunits, which are folded similarly, and in contrast also to the heme-free chains when bound together in dimeric apohemoglobin (a°,B°). Moreover, it was shown that, once separated, the globin chains do not readily recombine at neutral pH and accessible temperatures to form apohemoglobin. The second paper demonstrated that if either chain contains a heme, then the complementary chains do specifically recombine. During such a recombination, the disordered a-globin is refolded to the conformation it possesses in apohemoglobin. The coupled binding and refolding was termed an alloplex interactiont, to emphasizethe extensive refolding induced by a neighboring subunit.In future papers on this subject, the kinetics and mechanism of the alloplex interaction and the equilibrium of the HFa and HFB intermediates will be reported. The present paper shows that human hemoglobin can be reconstituted from separated globin chains and hemin by two different routes and that the reconstituted hemoglobin is similar or identical to native hemoglobin with respect to cooperativity in oxygen binding, the Bohr effect, and response to the allosteric effector 2,3-diphosphoglycerate (P-glycerate).In the course of our work on this problem, we found it necessary to modify several of our previous...

We have used a custom-built ultrasound velocimeter to carry out high-precision velocity measurements of reverse micelle solutions, made of ionic (AOT) and nonionic (C12E4) surfactants in oil, as a function of water concentration. We show that the observed velocity variation as a function of increasing water concentration differs from the characteristics of the surfactant polar headgroups. The complex profile of compressibility curves obtained from velocity and densimetric measurements can be accounted for by the relation existing between the surface polar headgroup of each surfactant and the number of interacting water molecules. At the highest water concentration, the compressibility parameters obtained are different from those reported for “bulk” water and reflect the peculiar properties of confined water.