Activity coefficients of salts in highly concentrated protein solutions: I. Alkali chlorides in isoionic bovine serum albumin solutions

In order to understand the thermodynamic state of simple salts in living cells, the mean activity coefficients of LiCl, NaCl, KC1, RbCl, CsCl were determined in concentrated isoionic bovine serum albumin (BSA) solutions by use of the EMF method with ion exchange membrane electrodes. The protein concentration range extended up to 22 wt %, whereas the salt concentration was kept constant at 0.1 mole per kilogram water. These solutions may be regarded as crude but appropriate model systems for the cytoplasm of cells as far as type and magnitude of the macromolecular component influence on the chemical potential of the salts is concerned. The mean stoichiometric activity coefficients of the alkali chlorides in the isoionic BSA solutions decreased linearly with the protein molality; this decrease, however, did not exceed ca. 10% compared with the pure 0.1 molal salt solutions. Only very small differences in the behaviour of the different alkali chlorides were observed. The results may be interpreted by the superposition of the effects of specific Cl^? ion binding to BSA and BSA bound “non-solvent” water with probably electrostatic long range interactions of the BSA(Cl^?)_v polyions with the salt ions in solution. The resulting mean activity coefficients, corrected for ion binding and non-solvent water, showed a very slight linear dependence on the protein concentration. The departure from the value in the pure 0.1 molal salt solutions did not exceed ± 2%.     

Exploring the differences and similarities between urea and thermally driven denaturation of bovine serum albumin: intermolecular forces and solvation preferences

The interactions of bovine serum albumin (BSA) with urea/water were investigated by computer simulation. It was revealed that the BSA-hydrophobic residues in urea solutions favored contact with urea more than with water. Energy decomposition analysis showed that van der Waals energy was the dominant driving force behind urea affinity for hydrophobic residues, whereas coulombic attraction was largely responsible for water affinity for these residues. Meanwhile, urea–BSA hydrogen bond energies were found to be weaker than water–BSA hydrogen bond energies. The greater strength of water–BSA hydrogen bonds than urea–BSA hydrogen bonds, and the opposing preferential interaction between the BSA and urea suggest that disruption of hydrophobic interaction predominates urea–protein denaturation. In pure water, hydrophobic residues showed aggregation tendencies at 323 K, suggesting an increase in hydrophobicity, while at 353 K the residues were partly denatured due to loss of hydrogen bonds; thus, disruption of hydrophobic interactions appeared to contribute less to thermal denaturation.     

π-Cation interactions as the origin of the weak absorption at 532 nm observed in tryptophan-containing polypeptides

We have previously reported that bovine serum albumin (BSA) and other proteins that do not contain prosthetic groups exhibited a weak light absorption in the visible, only detectable by pulsed laser-induced optoacoustic spectroscopy (LIOAS). Human serum albumin (HSA) exhibited signals 25% higher than those observed with BSA. Signals comparable to those obtained with BSA were observed with poly(L-Trp, L-Lys), poly(L-Trp, L-Arg) or poly(L-Trp, L-Orn) at pH 7.0. No signals were obtained when tryptophan was replaced by other amino acids or when free tryptophan or the tripeptide Lys-Trp-Lys was assayed (pH 7.0). Tryptophan in HCl 5 N produced LIOAS signals similar to those produced by tryptophan-containing copolymers. Moreover, the absorption peak could be observed in a UV-VIS spectrophotometer. Therefore, the LIOAS signals obtained with BSA, HSA, and tryptophan-containing random copolymers may be attributed to a new transition of the indole moiety of their tryptophan residues when "protonated". Tryptophan residues of proteins are known to participate in π-cation interactions, which are important in protein stability and function. As a matter of fact, HSA and BSA contain an internal tryptophan in close proximity to lysine and arginine residues and therefore suitable for π-cation interactions. The strength of this type of interaction strongly depends on distances and relative orientations of both amino acid residues. Accordingly, these interactions should be highly sensitive to conformational changes. Based on preliminary results that have shown that LIOAS signal at 532 nm depended on the aggregation state of BSA and/or on the oxidation state of its Cys-34, we postulate that the LIOAS signal observed with proteins and tryptophan-containing polypeptides are related to Trp-Lys or Trp-Arg interactions and that the intensity of the signal depends on the strength of such interactions.     

Ionic-strength-dependent effects in protein folding: analysis of rate equilibrium free-energy relationships and their interpretation

The traditional approach to studying protein folding involves applying a perturbation, usually denaturant or mutation, and determining the effect upon the free energy of folding, DeltaG0, and the activation free energy, DeltaG(not equal). Data collected as a function of the perturbation can be used to construct rate equilibrium free-energy relationships, which report on the development of interactions in the transition state for folding. We examine the use of the ionic-strength-dependent rate equilibrium free-energy relationship in protein folding using the N-terminal domain of L9, a small alpha-beta protein, as a model system. Folding is two-state for the range of ionic strength examined, 0.045-1.52 M. The plot of DeltaG(not equal) versus DeltaG0 is linear (r2= 0.918), with a slope equal to 0.45. The relatively low value of the slope indicates that the ionic-strength-dependent interactions are modestly developed in the transition state. The slope is, however, greater than that of a plot of DeltaG(not equal) versus DeltaG0 constructed by varying pH, thus demonstrating directly that ionic-strength-dependent studies probe more than simple electrostatic interactions. Potential transition movement was probed by analysis of the denaturant, ionic strength cross-interaction parameters. The values are small but nonzero and positive, suggesting a small shift of the transition state toward the native state as the protein is destabilized, i.e., Hammond behavior. The complications that arise in the interpretation of ionic-strength-dependent rate equilibrium free-energy relationships are discussed, and it is concluded that the ionic-strength-dependent studies do not provide a reliable indicator of the role of electrostatic interactions. Complications include incomplete screening of electrostatic interactions, specific ion binding, Hofmeister effects, and the potential presence of electrostatic interactions in the denatured state ensemble.     

Effects of arginine on heat-induced aggregation of concentrated protein solutions

Arginine is one of the commonly used additives to enhance refolding yield of proteins, to suppress aggregation of proteins, and to increase solubility of proteins, and yet the molecular interactions that contribute to the role of arginine are unclear. Here, we present experiments, using bovine serum albumin (BSA), lysozyme (LYZ), and β-lactoglobulin (BLG) as model proteins, to show that arginine can enhance heat-induced aggregation of concentrated protein solutions, contrary to the conventional belief that arginine is a universal suppressor of aggregation. Results show that the enhancement in aggregation is caused only for BSA and BLG, but not for LYZ, indicating that arginine's preferential interactions with certain residues over others could determine the effect of the additive on aggregation. We use this previously unrecognized behavior of arginine, in combination with density functional theory calculations, to identify the molecular-level interactions of arginine with various residues that determine arginine's role as an enhancer or suppressor of aggregation of proteins. The experimental and computational results suggest that the guanidinium group of arginine promotes aggregation through the hydrogen-bond-based bridging interactions with the acidic residues of a protein, whereas the binding of the guanidinium group to aromatic residues (aggregation-prone) contributes to the stability and solubilization of the proteins. The approach, we describe here, can be used to select suitable additives to stabilize a protein solution at high concentrations based on an analysis of the amino acid content of the protein.     

Spectroscopic study of the interaction of gossypol with bovine serum albumin

The interaction of gossypol with bovine serum albumin (BSA) at pH 7.6 in 0.02 M borax-borate buffer has been followed by circular dichroism (CD) and difference spectroscopy. From the extrinsic CD band at 390 nm, a binding constant of 2.7 X 10(3) M-1 was calculated. At 54 degrees the induced CD spectrum was abolished, suggesting that the interaction is not favoured at that temperature. The effect of various solvents and salts on the interaction has been followed by difference spectroscopy. The modification of epsilon-amino groups of lysine did not affect the interaction. Binding of gossypol to BSA does not cause a change in its secondary structure or sedimentation coefficient.     

Interference of some aqueous two-phase system phase-forming components in protein determination by the Bradford method

The interference of some specific aqueous two-phase system (ATPS) phase-forming components in bovine serum albumin (BSA) determination by the Bradford method was investigated. For this purpose, calibration curves were obtained for BSA in the presence of different concentrations of salts and polymers. A total of 19 salts [Na₂SO₄, (NH₄)₂SO₄, MgSO₄, LiSO₄, Na₂HPO₄, sodium phosphate buffer (pH 7.0), NaH₂PO₄, K₂HPO₄, potassium phosphate buffer (pH 7.0), KH₂PO₄, C₆H₈O₇, Na₃C₆H₅O₇, KCHO₂, NaCHO₂, NaCO₃, NaHCO₃, C₂H₄O₂, sodium acetate buffer (pH 4.5), and NaC₂H₃O₂] and 7 polymers [PEG 4000, PEG 8000, PEG 20000, UCON 3900, Ficoll 70000, PES 100000, and PVP 40000] were tested, and each calibration curve was compared with the one obtained for BSA in water. Some concentrations of salts and polymers had considerable effect in the BSA calibration curve. Carbonate salts were responsible for the highest salt interference, whereas citric and acetic acids did not produce interference even in the maximum concentration level tested (5 wt%). Among the polymers, UCON gave the highest interference, whereas Ficoll did not produce interference when used in concentrations up to 10 wt%. It was concluded that a convenient dilution of the samples prior to the protein quantification is needed to ensure no significant interference from ATPS phase-forming constituents.     

Formaldehyde-mediated aggregation of protein antigens: comparison of untreated and formalinized model antigens

A formaldehyde-mediated aggregation pathway (FMAP) is suggested as being primarily responsible for the aggregation of lyophilized tetanus toxoid (TT; a formalinized antigen) in the presence of moisture. The general occurrence of the FMAP was examined by using bovine serum albumin (BSA) and ribonuclease A (RNase) as model antigens; both protein antigens were formalinized according to a method commonly used to detoxify bacterial toxins. To clearly delineate the FMAP from other aggregation mechanisms, the aggregation kinetics and mechanism of both unmodified antigens (BSA and RNase) and formalinized antigens (f-BSA and f-RNase) were evaluated. We report that formaldehyde treatment introduces more rapid and extensive aggregation in antigens under conditions that favor the FMAP (i.e., 80% relative humidity and 37 degrees C). Consistent with formaldehyde-mediated crosslinking, f-antigen aggregates were covalent and non-disulfide-bonded, whereas BSA aggregates were disulfide-linked and RNase even did not aggregate under the same conditions. Coincorporation of amino acids (histidine and lysine), which strongly interact with formaldehyde, as well as prior antigen reduction with cyanoborohydride, significantly inhibited f-BSA aggregation, but showed no selective effect on BSA aggregation. Mechanistic analysis of f-BSA aggregates, inhibition studies, and similar reactivity of f-BSA with TT all confirmed the existence of the FMAP at moisture levels intermediate between the dry and solution state. This study demonstrates the potential for covalent reactions between formalinized protein antigens and neighboring chemical or biochemical species even after formalinization, and provides a general approach to inhibit the FMAP.     

Complex formation of bovine serum albumin with a poly(ethylene glycol) lipid conjugate

In this work, we report the formation of complexes by self-assembly of bovine serum albumin (BSA) with a poly(ethylene glycol) lipid conjugate (PEG2000-PE) in phosphate saline buffer solution (pH 7.4). Three different sets of samples have been studied. The BSA concentration remained fixed (1, 0.01, or 0.001 wt % BSA) within each set of samples, while the PEG2000-PE concentration was varied. Dynamic light scattering (DLS), rheology, and small-angle X-ray scattering (SAXS) were used to study samples with 1 wt % BSA. DLS showed that BSA/PEG2000-PE aggregates have a size intermediate between a BSA monomer and a PEG2000-PE micelle. Rheology suggested that BSA/PEG2000-PE complexes might be surrounded by a relatively compact PEG-lipid shell, while SAXS results showed that depletion forces do not take an important role in the stabilization of the complexes. Samples containing 0.01 wt % BSA were studied by circular dichroism (CD) and ultraviolet fluorescence spectroscopy (UV). UV results showed that at low concentrations of PEG-lipid, PEG2000-PE binds to tryptophan (Trp) groups in BSA, while at high concentrations of PEG-lipid the Trp groups are exposed to water. CD results showed that changes in Trp environment take place with a minimal variation of the BSA secondary structure elements. Finally, samples containing 0.001 wt % BSA were studied by zeta-potential experiments. Results showed that steric interactions might play an important role in the stabilization of the BSA/PEG2000-PE complexes.     

Osmotically unresponsive water fraction on proteins: non-ideal osmotic pressure of bovine serum albumin as a function of pH and salt concentration

How much does protein-associated water differ in colligative properties (freezing point, boiling point, vapor pressure and osmotic behavior) from pure bulk water? This question was approached by studying the globular protein bovine serum albumin (BSA), using changes in pH and salt concentration to alter its native structural conformation and state of aggregation. BSA osmotic pressure was investigated experimentally and analyzed using the molecular model of Fullerton et al. [Biochem Cell Biol 1992;70(12):1325]. Analysis yielded both the extent of osmotically unresponsive water (OUW) and the effective molecular weight values of the membrane-impermeable BSA solute. Manipulation of BSA conformation and aggregation by membrane-penetrating cosolutes show that alterations in pH and salt concentration change the amount of bulk water that escapes into BSA from a minimum of 1.4 to a maximum of 11.7 g water per g dry mass BSA.     

Covalent complexes of albumin with serotonin, ketanserin and lysergic acid antagonize the activity of serotonin in human platelets

Covalent conjugates of bovine serum albumin (BSA) and 5-HT, ketanserin or d-lysergic acid were synthesized and characterized by polyacrylamide gel electrophoresis, whole blood clearance experiments in mice and aggregation studies with human platelets. Using the standard synthesis procedure, each mol of BSA bound 13.4 mol of [3H]5-HT. Derivatization did not cause significant protein aggregation as determined by electrophoresis. All three conjugates antagonized the ability of 5-HT to amplify aggregation caused by low concentrations of ADP. The antagonist activity of each conjugate was concentration dependent; 2.6 microM 5-HT-BSA completely inhibited the aggregation caused by 13 microM 5-HT. None of the BSA drug conjugates, including 5-HT-BSA, amplified platelet aggregation caused by ADP in the absence of 5-HT. Aggregation by ristocetin, collagen, epinephrine or ADP alone was not significantly affected by the conjugates. Whole blood elimination experiments in mice demonstrated that the three conjugates and underivatized BSA are equally stable in the circulation. These prototypic 5-HT drug-protein conjugates may be useful for probing 5-HT2 receptor-ligand interactions in human platelets.     

How does dextran sulfate prevent heat induced aggregation of protein? The mechanism and its limitation as aggregation inhibitor

The effect of dextran sulfate on protein aggregation was investigated to provide the clues of its biochemical mechanism. The interaction between dextran sulfate and BSA varied with the pH values of the solution, which led to the different extent of aggregation prevention by dextran sulfate. Light scattering data with thermal scan showed that dextran sulfate suppressed BSA aggregation at pH 5.1 and pH 6.2, while it had no effect at pH 7.5. Isothermal titration calorimetric analysis suggested that the pH dependency of the role of dextran sulfate on BSA aggregation would be related to the difference in the mode of BSA-dextran sulfate complex formation. Isothermal titration calorimetric analysis at pH 6.2 indicated that dextran sulfate did not bind to native BSA at this pH, but interacted with partially unfolded BSA. While stabilizing native form of protein by the complex formation has been suggested as the suitable mechanism of preventing aggregation, our observation of conformational changes by circular dichroism spectroscopy showed that strong electrostatic interaction between dextran sulfate and BSA rather facilitated the denaturation of BSA. Combining the data from isothermal titration calorimetry, circular dichroism, and dynamic light scattering, we found that the complex formation of the intermediate state of denatured BSA with dextran sulfate is a prerequisite to suppress the aggregation by preventing further oligomerization/aggregation process of denatured protein.     

Modulation of the bilayer to hexagonal phase transition and solvation of phosphatidylethanolamines in aqueous salt solutions

Several salts affect the temperature of the bilayer to hexagonal phase transition of phosphatidylethanolamines. Their effects are dependent on the anion as well as the cation of the salt. Salt effects on this transition can be explained by preferential hydration and ion binding. Those salts which are excluded from the solvation sphere of the membrane promote hexagonal phase formation. For example, Na2SO4 promotes preferential hydration and is a hexagonal phase promoter while NaSCN does not do this and is a bilayer stabilizer. Unlike amphiphiles and hydrocarbons, salts can shift the bilayer to hexagonal phase transition temperature without altering the cooperativity of the transition. The effect of these salts on the gel to liquid-crystal transition is opposite to their effect on the bilayer to hexagonal phase transition. We also find that MnCl2 markedly raises the gel to liquid-crystal transition temperature. This effect is due to binding of the cation to the membrane surface. The effect is reduced with MnSO4 because of preferential hydration. Our results demonstrate that the nature of the anion as well as the cation can alter the effect of salts on lipid phase transition properties. The observed effects can be explained as resulting from preferential hydration and ion binding.     

Adsorption of BSA on QAE-dextran: equilibria

Equilibrium isotherms for adsorption of bovine serum albumin (BSA) on a strong-base (QAE) dextran-type ion exchanger have been determined experimentally. They were not affected by the initial concentration of BSA but were affected by pH considerably. They were correlated by the Langmuir equation when pH >/= 5.05 and by the Freundlich equation of pH 4.8, which is close to pl approximately 4.8 of BSA. The contribution of ion exchange to adsorption of BSA on the ion exchanger was determined experimentally. The maximum amounts of inorganic anion exchanged for BSA were 1% and 0.4% of the exchange capacity of the ion exchanger at pH 6.9, respectively. Since the effect of the ion exchange on the adsorption appeared small, BSA may be adsorbed mainly by electrostatic attraction when pH >/= 5.05 and by hydrophobic interaction or hydrogen bonding at pH 4.8. When NaCl coexisted in the solution, the shape of the isotherm was similar to the Langmuir isotherm, but it is shifted to the right. When the concentration of NaCl was 0.2 mol/dm(3), BsA was not adsorbed on the resin. When BSA was dissolved in pure water, the saturation capacity of BSA on HPO(4) (2-),-orm resin was about 2 times larger than that for adsorption from the solution with buffer (pH 6.9 and 8.79). The saturation capacity for adsorption of BSA in pure water on HPO(4) (2-) + H(2)O(4) (-)-from resin was much smaller than that from the solution with buffer. The isotherms for univalent Cl(-)-and H(2)PO(4) (-)-form resin was peculiar; that is, the amount of BSA adsorbed decreased with increasing the liquid-phase equilibrium concentration of BSA. (c) 1993 John Wiley & Sons, Inc.     

Intrinsic electric fields and proton diffusion in immobilized protein membranes. Effects of electrolytes and buffers.

We present a theory for proton diffusion through an immobilized protein membrane perfused with an electrolyte and a buffer. Using a Nernst-Planck equation for each species and assuming local charge neutrality, we obtain two coupled nonlinear diffusion equations with new diffusion coefficients dependent on the concentration of all species, the diffusion constants or mobilities of the buffers and salts, the pH-derivative of the titration curves of the mobile buffer and the immobilized protein, and the derivative with respect to ionic strength of the protein titration curve. Transient time scales are locally pH-dependent because of protonation-deprotonation reactions with the fixed protein and are ionic strength-dependent because salts provide charge carriers to shield internal electric fields. Intrinsic electric fields arise proportional to the gradient of an "effective" charge concentration. The field may reverse locally if buffer concentrations are large (greater to or equal to 0.1 M) and if the diffusivity of the electrolyte species is sufficiently small. The "ideal" electrolyte case (where each species has the same diffusivity) reduces to a simple form. We apply these theoretical considerations to membranes composed of papain and bovine serum albumin (BSA) and show that intrinsic electric fields greatly enhance the mobility of protons when the ionic strength of the salts is smaller than 0.1 M. These results are consistent with experiments where pH changes are observed to depend strongly on buffer, salt, and proton concentrations in baths adjacent to the membranes.     

Influence of cellular environment on toxicity of nitroheterocycles.

The preferential sensitivity of hypoxic cells to nitroheteroxycles is thought to result from the actions of toxic intermediates of drug reduction produced under hypoxic conditions. However, a lack of oxygen also alters the biochemical state of the cell and may indirectly enhance the sensitivity, of hypoxic cells to these drugs. This hypothesis was tested by 'conditioning' mouse L-929 cells in oxygen-free buffer, then exposing the cells to nitrofurazone under both aerobic and anaerobic conditions. After conditioning, the rate of cell inactivation by nitrofurazone was equal in air or nitrogen-equilibrated buffer. Pretreatment of cells in 1 muM rotenone or 0.5 mM 2,4-dinitrophenol for one hour under aerobic conditions increased the sensitivity of the cells to nitrofurazone under aerobic conditions. Similar rates of cell killing were obtained when mouse L-cells were heated in buffer for 30 min at 43 degrees before incubation with nitrofurazone in either air or nitrogen. Also, incubation of cells with nitrofurazone in the presence of 0.1% glucose, or at a cell density less than 10(5) cells/ml significantly enhanced cell killing, especially under aerobic conditions. Thus, the intracellular state of the cell, manipulated by altering the cellular environment, influenced the cellular sensitivity to nitrofurazone. Similar results were not, however, obtained with the nitroimidazoles, dimetronidazole and misonidazole; pretreatment for 2 h in buffer under anaerobic conditions did not increase the sensitivity of L cells to subsequent drug treatment in air-equilibrated buffer.     

Effect of salts on the properties of aqueous sugar systems, in relation to biomaterial stabilization. 1. Water sorption behavior and ice crystallization/melting

Trehalose and sucrose, two sugars that are involved in the protection of living organisms under extreme conditions, and their mixtures with salts were employed to prepare supercooled or freeze-dried glassy systems. The objective of the present work was to explore the effects of different salts on water sorption, glass transition temperature (T(g)), and formation and melting of ice in aqueous sugar systems. In the sugar-salt mixtures, water adsorption was higher than expected on the basis of the water uptake by each pure component. In systems with a reduced mass fraction of water (w less-than-or-equal 0.4), salts delayed water crystallization, probably due to ion-water interactions. In systems where > 0.6, water crystallization could be explained by the known colligative properties of the solutes. The glass transition temperature of the maximally concentrated matrix (T(g)') was decreased by the presence of salts. However, the actual T(g) values of the systems were not modified. Thus, the effect of salts on sorption behavior and formation of ice may reflect dynamic water-salt-sugar interactions which take place at a molecular level and are related to the charge/mass ratio of the cation present without affecting supramolecular or macroscopic properties.     

Combined mutation of catalytic glutamate residues in the two nucleotide binding domains of P-glycoprotein generates a conformation that binds ATP and ADP tightly

Combined mutation of "catalytic carboxylates" in both nucleotide binding domains (NBDs) of P-glycoprotein generates a conformation capable of tight binding of 8-azido-ADP (Sauna, Z. E., Müller, M., Peng, X. H., and Ambudkar, S. V. (2002) Biochemistry 41, 13989-14000). Here we characterized this conformation using pure mouse MDR3 P-glycoprotein and natural MgATP and MgADP. Mutants E552A/E1197A, E552Q/E1197Q, E552D/E1197D, and E552K/E1197K had low but real ATPase activity in the order Ala > Gln > Asp > Lys, emphasizing the requirement for Glu stereochemistry. Mutant E552A/E1197A bound MgATP and MgADP (1 mol/mol) with K(d) 9.2 and 92 microm, showed strong temperature sensitivity of MgATP binding and equal dissociation rates for MgATP and MgADP. With MgATP as the added ligand, 80% of bound nucleotide was in the form of ATP. None of these parameters was vanadate-sensitive. The other mutants showed lower stoichiometry of MgATP and MgADP binding, in the order Ala > Gln > Asp > Lys. We conclude that the E552A/E1197A mutation arrests the enzyme in a conformation, likely a stabilized NBD dimer, which occludes nucleotide, shows preferential binding of ATP, does not progress to a normal vanadate-sensitive transition state, but hydrolyzes ATP and releases ADP slowly. Impairment of turnover is primarily due to inability to form the normal transition state rather than to slow ADP release. The Gln, Asp, and Lys mutants are less effective at stabilizing the occluded nucleotide, putative dimeric NBD, conformation. We envisage that in wild-type the occluded nucleotide conformation occurs transiently after MgATP binds to both NBDs with associated dimerization, and before progression to the transition state.     

Thermal induced conformational changes involved in the aggregation pathways of beta-lactoglobulin

Aggregation of proteins appears to be associated most often with conformational and structural changes that lead to exposure of some apolar residues. Depending on the native structure of the protein in exam, aggregation is a process that involves different mechanisms, whose time of occurrence and interplay can depend upon temperature. To single out information about the multistages of the aggregation pathway, here we investigate the thermally induced conformational and structural changes of the beta-lactoglobulin (BLG). The experimental approach consists in studying steady-state fluorescence spectra of intrinsic chromophores, two tryptophans, and Anylino-Naphthalene-Sulfonate dye (ANS) molecular probe. This technique revealed to be particularly suitable in investigating samples in the low concentration range and at the initial steps of the aggregation process. The Rayleigh scattering of the exciting light follows the growth of the intermolecular interactions at the same time. Complementary information is also obtained by circular dichroism (CD) measurements on samples in the same experimental conditions. The obtained data indicate a well-defined interconversion between quaternary, ternary and secondary structures, together with conformational rearrangements driven by hydrophobic interactions and intermolecular bonds. The results are also discussed in comparison with similar studies on BSA aggregation.