Domain II + III of bovine serum albumin: Isolation and its characterization

Some properties of a fragment of bovine serum albumin containing residues 184–582 of the protein sequence, produced by cyanogen bromide cleavage, have been reported. Urea-induced difference spectra of the fragment showed considerable exposure of aromatic chromophores by 8 M urea. Reversible unfolding of the fragment by urea, as followed by difference spectral measurements at 30°C, pH 7.0, occurred in two distinct steps involving at least 3 major conformational states, namely the native (N), intermediate (X) and completely denatured (D) states. The co-operativity values for the two transitions, N⇌X and X⇌Dwere found to be 4.0 and 16.4, respectively. Analysis of the data on bilirubin binding to bovine serum albumin and its fragment suggested that the fragment retains significant amount of its native structure. However, hydrodynamic parameters such as Stokes radius (3.f14 nm), diffusion coefficient (6.98 × 10⁻⁷cm²/s) and frictional ratio (1.32) obtained by analytical gel chromatography as well as intrinsic viscosity (4.31 ml/g) indicates some asymmetry in the fragment molecule.     

The Limitations of an Exclusively Colloidal View of Protein Solution Hydrodynamics and Rheology

Proteins are complex macromolecules with dynamic conformations. They are charged like colloids, but unlike colloids, charge is heterogeneously distributed on their surfaces. Here we overturn entrenched doctrine that uncritically treats bovine serum albumin (BSA) as a colloidal hard sphere by elucidating the complex pH and surface hydration-dependence of solution viscosity. We measure the infinite shear viscosity of buffered BSA solutions in a parameter space chosen to tune competing long-range repulsions and short-range attractions (2 mg/mL ≤ [BSA] ≤ 500 mg/mL and 3.0 ≤ pH ≤ 7.4). We account for surface hydration through partial specific volume to define volume fraction and determine that the pH-dependent BSA intrinsic viscosity never equals the classical hard sphere result (2.5). We attempt to fit our data to the colloidal rheology models of Russel, Saville, and Schowalter (RSS) and Krieger-Dougherty (KD), which are each routinely and successfully applied to uniformly charged suspensions and to hard-sphere suspensions, respectively. We discover that the RSS model accurately describes our data at pH 3.0, 4.0, and 5.0, but fails at pH 6.0 and 7.4, due to steeply rising solution viscosity at high concentration. When we implement the KD model with the maximum packing volume fraction as the sole floating parameter while holding the intrinsic viscosity constant, we conclude that the model only succeeds at pH 6.0 and 7.4. These findings lead us to define a minimal framework for models of crowded protein solution viscosity wherein critical protein-specific attributes (namely, conformation, surface hydration, and surface charge distribution) are addressed.     

A correlation between changes in conformation and molecular properties of bovine serum albumin upon succinylation

Using succinic anhydride, six succinylated derivatives of bovine serum albumin having percent modification in the range of 23-87% were prepared and their physicochemical and immunological properties were studied. Measurements of Stokes radius, frictional ratio, UV spectra, solvent perturbation, solubility, and immunological cross-reactivity against anti-bovine serum albumin antiserum revealed that the protein undergoes gradual changes in its native conformation with increase in the degree of succinylation. These changes were less marked below 50% modification but became pronounced above 50% modification. However, even the maximally modified preparation (87%) contained a significant amount of folded structure. Interestingly, though the measurements of various molecular properties revealed significant changes in 23-49% modified preparations, the solubility parameters for these preparations which were obtained at high ionic strength were indistinguishable from those of the native protein. The various results taken together suggest that at lower degrees of chemical modification, the conformational changes were produced mainly because of an increase in electrostatic free energy, whereas at higher degrees of modification, steric hindrance in addition to the electrostatic factor seems to make a substantial contribution to the conformational changes in the modified proteins.     

The Limitations of an Exclusively Colloidal View of Protein Solution Hydrodynamics and Rheology

Proteins are complex macromolecules with dynamic conformations. They are charged like colloids, but unlike colloids, charge is heterogeneously distributed on their surfaces. Here we overturn entrenched doctrine that uncritically treats bovine serum albumin (BSA) as a colloidal hard sphere by elucidating the complex pH and surface hydration-dependence of solution viscosity. We measure the infinite shear viscosity of buffered BSA solutions in a parameter space chosen to tune competing long-range repulsions and short-range attractions (2 mg/mL ≤ [BSA] ≤ 500 mg/mL and 3.0 ≤ pH ≤ 7.4). We account for surface hydration through partial specific volume to define volume fraction and determine that the pH-dependent BSA intrinsic viscosity never equals the classical hard sphere result (2.5). We attempt to fit our data to the colloidal rheology models of Russel, Saville, and Schowalter (RSS) and Krieger-Dougherty (KD), which are each routinely and successfully applied to uniformly charged suspensions and to hard-sphere suspensions, respectively. We discover that the RSS model accurately describes our data at pH 3.0, 4.0, and 5.0, but fails at pH 6.0 and 7.4, due to steeply rising solution viscosity at high concentration. When we implement the KD model with the maximum packing volume fraction as the sole floating parameter while holding the intrinsic viscosity constant, we conclude that the model only succeeds at pH 6.0 and 7.4. These findings lead us to define a minimal framework for models of crowded protein solution viscosity wherein critical protein-specific attributes (namely, conformation, surface hydration, and surface charge distribution) are addressed.     

Anomalous salting-out behaviour of cyanogen bromide fragments of bovine serum albumin

The solubilities of bovine serum albumin and its two cyanogen bromide fragments comprising domain I and II+III of the protein in ammonium sulphate solution were studied at different pH and temperature and the salting-out parameters K_s and β were determined for the three proteins. The values of K_s and β obtained for the intact albumin at different pH were atypical of other globular proteins and were explained in terms of N-F transition and pH induced unfolding of the protein. The salting-out behaviour of the two fragments was, however, found to be significantly different from that of their parent molecule. In contrast to bovine serum albumin, the aqueous solubilities of the two fragments were highly dependent on temperature. Similarly, pH dependence of β for the two fragments was also different since it acquired a minimum value at about pH 4.0 as against its monotonic decrease with pH observed in intact albumin below pH 5.0. Anomalous salting-out behaviour of the two cyanogen bromide fragments has been attributed to the possible conformational changes that might occur during the course of their preparation under relatively harsher chemical conditions.     

Alginate as a support ligand for enhanced colloidal liquid aphron immobilization of proteins and drug delivery

Research within the field of colloidal liquid aphrons (CLAs) for enzyme immobilization has often used ionic surfactants for the retention of enzymes. Although these charged interactions allow for enhanced immobilization, they can often lead to denaturation of enzyme activity, and even release of the protein. Sodium alginate has been used in drug delivery applications due to its low toxicity and charged interactions that allow for encapsulation. Hence, alginate systems can be used as an alternative to ionic surfactants in CLA immobilization. This paper presents, for the first time, the use of sodium alginate as potential ligand for enhanced CLA immobilization. The use of five model proteins; lysozyme, bovine serum albumin, ovalbumin, insulin, and α-chymotrypsin, of various pIs and hydrophobicities, showed the relevance of electrostatic interactions in promoting binding with sodium alginate when the pH < pI, with 100% immobilization attributed to alginate incorporated CLAs over general nonionic formulations. Furthermore, above their pI, >80% protein recovery was observed, with activity and conformation comparable to their native counterparts. Finally, the use of proteolysis showed that as the degree of ionic bonding increased between the protein and sodium alginate, the degree of protease resistance decreased due to conformational changes experienced during binding.     

Thermodynamics of the temperature-induced unfolding of globular proteins.

The heat capacity, enthalpy, entropy, and Gibbs energy changes for the temperature-induced unfolding of 11 globular proteins of known three-dimensional structure have been obtained by microcalorimetric measurements. Their experimental values are compared to those we calculate from the change in solvent-accessible surface area between the native proteins and the extended polypeptide chain. We use proportionality coefficients for the transfer (hydration) of aliphatic, aromatic, and polar groups from gas phase to aqueous solution, we estimate vibrational effects, and we discuss the temperature dependence of each constituent of the thermodynamic functions. At 25 degrees C, stabilization of the native state of a globular protein is largely due to two favorable terms: the entropy of non-polar group hydration and the enthalpy of interactions within the protein. They compensate the unfavorable entropy change associated with these interactions (conformational entropy) and with vibrational effects. Due to the large heat capacity of nonpolar group hydration, its stabilizing contribution decreases quickly at higher temperatures, and the two unfavorable entropy terms take over, leading to temperature-induced unfolding.     

Fluorescence Spectroscopy as a Tool to Unravel the Dynamics of Protein Nanoparticle Formation by Liquid Antisolvent Precipitation

Protein-based particles are very promising colloidal systems for protection and controlled release applications in the food, cosmetics and pharmaceutical sector. One technique to produce these protein colloidal particles is liquid antisolvent precipitation (LAS). Despite the simplicity and versatility of LAS, not much is known about the protein conformational changes and interactions that are at the basis of the particle formation process. In this study, steady state fluorescence experiments using intrinsic fluorophores were evaluated as a tool to unravel the dynamics of the protein nanoparticle formation. Colloidal whey protein isolate and gliadin particles were produced by LAS. Changes in particle diameter (distribution), polydispersity index and photophysical properties of intrinsic fluorophores were monitored as a function of antisolvent concentration. By combining dynamic light scattering with photophysical data, a model of the changes occurring during particle formation and disintegration could be proposed. The results suggest that particle formation and disintegration are fully reversible processes during which the main changes in protein conformation (around the fluorescent probes) occur at the same antisolvent concentrations. In principle, steady state fluorescence measurements using intrinsic probes can indeed be used to effectively report on (part of the) conformational changes for both protein systems under study.     

Compressibility of protein transitions

We review the results of compressibility studies on proteins and low molecular weight compounds that model the hydration properties of these biopolymers. In particular, we present an analysis of compressibility changes accompanying conformational transitions of globular proteins. This analysis, in conjunction with experimental compressibility data on protein transitions, were used to define the changes in the hydration properties and intrinsic packing associated with native-to-molten globule, native-to-partially unfolded, and native-to-fully unfolded transitions of globular proteins. In addition, we discuss the molecular origins of predominantly positive changes in compressibility observed for pressure-induced denaturation transitions of globular proteins. Throughout this review, we emphasize the importance of compressibility data for characterizing protein transitions, while also describing how such data can be interpreted to gain insight into role that hydration and intrinsic packing play in modulating the stability of and recognition between proteins and other biologically important compounds.     

A contribution to the theory of biological staining based on the principles for structural organization of biological macromolecules

Biological staining is to a large degree explainable based on the principles governing folding and aggregation of macromolecules in aqueous solution. Most macromolecules are polyions, which, except for heteropolysaccharides, have a large proportion of nonpolar or only slightly polar residues. Because they are amphiphilic, they react in water by a complex set of hydrophobic interactions involving charged residues, nonpolar residues and water molecules. The hydrophobic interactions lead to complex folding systems or micelle-like structures. Dyes are amphiphilic molecules with a tendency to form micelles, but with limitations due to geometric constraints and charge repulsion. Macromolecules and dyes react with each other in aqueous solution following the same principles as for the structural organization of macromolecules, as in protein folding for example. Dye binding requires near contact between nonpolar groups in both the dye and macromolecule, and this is accomplished by choosing a pH at which the dye and macromolecule have opposite net charges. Charge attraction is insufficient for binding in most cases, but it is directive because it determines which macromolecules a given dye ion is able to contact. These considerations apply to the staining of globular (cytoplasmic) proteins and to nucleic acid staining. The staining mechanism is by hydrophobic interactions. Above approximately pH 3.5, DNA may also bind dyes by hydrophobic intercalation between the bases of the double helix; at lower pH the double helix opens and dye binding is as for RNA and globular proteins. Heteroglycans (mucins) have virtually no nonpolar groups, so nonpolar interactions are restricted to the dye molecules. Metachromatic staining of heteroglycans is due to hydrophobic bonding or micelle formation between the monovalent planar dye molecules aided by charge neutralization by the negatively charged heteroglycans. Alternatively, as the charge attraction increases with the number of closely placed charges, acidic heteroglycans may be stained by a polycation such as alcian blue or colloidal iron. For elastic fiber and collagen staining, actual hydrophobic interactions are less important and hydrogen bonding and simple nonpolar interactions play a major role. These macromolecules may therefore be stained using a nonaqueous alcoholic solution.     

A contribution to the theory of biological staining based on the principles for structural organization of biological macromolecules

Biological staining is to a large degree explainable based on the principles governing folding and aggregation of macromolecules in aqueous solution. Most macromolecules are polyions, which, except for heteropolysaccharides, have a large proportion of nonpolar or only slightly polar residues. Because they are amphiphilic, they react in water by a complex set of hydrophobic interactions involving charged residues, nonpolar residues and water molecules. The hydrophobic interactions lead to complex folding systems or micelle-like structures. Dyes are amphiphilic molecules with a tendency to form micelles, but with limitations due to geometric constraints and charge repulsion. Macromolecules and dyes react with each other in aqueous solution following the same principles as for the structural organization of macromolecules, as in protein folding for example. Dye binding requires near contact between nonpolar groups in both the dye and macromolecule, and this is accomplished by choosing a pH at which the dye and macromolecule have opposite net charges. Charge attraction is insufficient for binding in most cases, but it is directive because it determines which macromolecules a given dye ion is able to contact. These considerations apply to the staining of globular (cytoplasmic) proteins and to nucleic acid staining. The staining mechanism is by hydrophobic interactions. Above approximately pH 3.5, DNA may also bind dyes by hydrophobic intercalation between the bases of the double helix; at lower pH the double helix opens and dye binding is as for RNA and globular proteins. Heteroglycans (mucins) have virtually no nonpolar groups, so nonpolar interactions are restricted to the dye molecules. Metachromatic staining of heteroglycans is due to hydrophobic bonding or micelle formation between the monovalent planar dye molecules aided by charge neutralization by the negatively charged heteroglycans. Alternatively, as the charge attraction increases with the number of closely placed charges, acidic heteroglycans may be stained by a polycation such as alcian blue or colloidal iron. For elastic fiber and collagen staining, actual hydrophobic interactions are less important and hydrogen bonding and simple nonpolar interactions play a major role. These macromolecules may therefore be stained using a nonaqueous alcoholic solution.     

Conformational flexibility of alpha-lactalbumin related to its membrane binding capacity

Different folding states of the small, globular milk protein bovine alpha-lactalbumin (BLA) induced by the anionic surfactant sodium dodecylsulphate (SDS) have been examined by fluorescence spectroscopy, CD and NMR. The solution structure of the protein in the absence of SDS was also determined, indicating fluidity even under native conditions. BLA is partly denatured to a molten globule (MG)-like state by micromolar concentrations of SDS, and the transitions from native to MG-like state are dependent on pH, the protein being more sensitive to the surfactant at pH 6.5. As indicated by measurements of the intrinsic emission fluorescence, the tertiary structure disappears at lower concentrations of SDS than most of the secondary structure, as estimated from CD data. The MG-like state induced by low concentrations of SDS is not observable by NMR, and is probably fluctuating and/or aggregating. At higher concentrations of SDS above the critic concentration of micelles, an NMR-observable state reappears. This micelle-associated conformer was partially assigned, and found to bear strong resemblance to the acid-tri-fluoroethanol state, retaining weakened versions of the A and C helix of native BLA. We discuss the results in terms of the inherent flexibility of the protein, and its ability to form multiple folding states and to bind to membranes. Also, we propose that proteins with stable MG-like conformers can have these states stabilized by low levels of compounds with surfactant properties in vivo.     

Determination of the axial ratio of globular proteins in aqueous solution using viscometric measurements

The paper presents the results of viscosity determinations on aqueous solutions of several globular proteins in a wide range of concentrations. On the basis of these measurements a general formula connecting the relative viscosity with concentration and axial ratio of the dissolved proteins was established. By applying the formula the axial ratios of bovine γ-globulin and horse albumin molecules were calculated.     

EPR study of non-covalent spin labeled serum albumin and hemoglobin

Electron Paramagnetic Resonance (EPR) was used to investigate the Tempyo spin label (3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy) as a report group for the interactions and the conformational changes of lyophilized bovine serum albumin (BSA) and bovine hemoglobin (BH), as function of pH values in the range 2.5-11. The EPR spectra are similar with those of other non-covalently spin label porphyrins in frozen solution at very low temperatures. This behavior indicated a possible spin-spin interaction between the hemic iron and the nitroxide group. The changes in the EPR spectra as function of the pH are discussed in terms of conformational changes of the proteins. Spectral simulations and magnetic EPR parameters reveal the following: (i) one single paramagnetic species, with Gaussian line shape, was used for the best fits of experimental spectra in the case of serum albumin samples; and (ii) a weighted sum of Lorentzian and Gaussian line shape in the case of hemoglobin samples. The representation of correlation time vs. pH, reveals a dependence of degree of immobilization of spin label on the conformational changes of proteins in acidic and basic environment.     

Effects of disulfide bonds on compactness of protein molecules revealed by volume, compressibility, and expansibility changes during reduction

To elucidate the effects of disulfide bonds on the compactness of protein molecules, the partial specific volume (v(o)) and coefficients of adiabatic compressibility (beta(s)(o)) and thermal expansibility (alpha) of five globular proteins (ovalbumin, beta-lactoglobulin, lysozyme, ribonuclease A, and bovine serum albumin) were measured in aqueous solutions with pH values of 7 and 2 at 25 degrees C when their disulfide bonds were totally reduced by carboxamidomethylation. Circular dichroism and fluorescence spectra show that the secondary and tertiary structures are partly disrupted by reduction, depending on the number of disulfide bonds in the proteins and the pH of the medium. The conformational changes are accompanied by decreases in v(o) and beta(s)(o) and by an increase in alpha, indicating that reduction decreases the internal cavity and increases surface hydration. The beta(s)(o) values of native or oxidized proteins decrease, and the effects of reduction on the volumetric parameters become more significant as the number of disulfide bonds increases and as they are formed over a larger distance in the primary structure. These results demonstrate that disulfide bonds play an important role, mainly via entropic forces, in the three-dimensional structure and compactness of protein molecules.     

Circular dichroism studies on conformational changes in protein molecules upon adsorption on ultrafine polystyrene particles

The conformational changes in well-characterized model proteins [bovine ribonuclease A (RNase A), horseradish peroxidase, sperm-whole myoglobin, human hemoglobin, and bovine serum albumin (BSA)] upon adsorption on ultrafine polystyrene (PS) particles have been studied using circular dichroism (CD) spectroscopy. These proteins were chosen with special attention to molecular flexibility. The ultrafine PS particles were negatively charged and have average diameters of 20 or 30 nm. Utilization of these ultrafine PS particles makes it possible to apply the CD technique to determine the secondary structure of proteins adsorbed on the PS surface. Effects of protein properties and adsorption conditions on the extent of the changes in the secondary structure of protein molecules upon adsorption on ultrafine PS particles were studied. The CD spectrum changes upon adsorption were significant in the "soft" protein molecules (myoglobin, hemoglobin, and BSA), while they were insingnificant in the "rigid" proteins (RNase A and peroxidase). The soft proteins sustained a marked decrease in alpha-helix content upon adsorption. Moreover, the native alpha-helix content, which is given as the percentage of the alpha-helix content in the free proteins, of adsorbed BSA was found to decrease with decreasing pH and increase with increasing adsorbed amount. These observations confirm some well-known hypotheses for the confirmational chages in protein molecules upon adsorption. (c) 1992 John Wiley & Sons, Inc.     

Stability of globular proteins in H2O and D2O

In several experimental techniques D2O rather then H2O is often used as a solvent for proteins. Concerning the influence of the solvent on the stability of the proteins, contradicting results have been reported in literature. In this paper the influence of H2O-D2O solvent substitution on the stability of globular protein structure is determined in a systematic way. The differential scanning calorimetry technique is applied to allow for a thermodynamic analysis of two types of globular proteins: hen's egg lysozyme (LSZ) with relatively strong internal cohesion ("hard" globular protein) and bovine serum albumin (BSA), which is known for its conformational adaptability ("soft" globular protein). Both proteins tend to be more stable in D2O compared to H2O. We explain the increase of protein stability in D2O by the observation that D2O is a poorer solvent for nonpolar amino acids than H2O, implying that the hydrophobic effect is larger in D2O. In case of BSA the transitions between different isomeric forms, at low pH values the Nm and F forms, and at higher pH values Nm and B, were observed by the presence of a supplementary peak in the DSC thermogram. It appears that the pH-range for which the Nm form is the preferred one is wider in D2O than in H2O.     

Characterization of highly concentrated antibody solution - A toolbox for the description of protein long-term solution stability

High protein titers are gaining importance in biopharmaceutical industry. A major challenge in the development of highly concentrated mAb solutions is their long-term stability and often incalculable viscosity. The complexity of the molecule itself, as well as the various molecular interactions, make it difficult to describe their solution behavior. To study the formulation stability, long- and short-range interactions and the formation of complex network structures have to be taken into account. For a better understanding of highly concentrated solutions, we combined established and novel analytical tools to characterize the effect of solution properties on the stability of highly concentrated mAb formulations. In this study, monoclonal antibody solutions in a concentration range of 50–200 mg/ml at pH 5–9 with and without glycine, PEG4000, and Na₂SO₄ were analyzed. To determine the monomer content, analytical size-exclusion chromatography runs were performed. ζ-potential measurements were conducted to analyze the electrophoretic properties in different solutions. The melting and aggregation temperatures were determined with the help of fluorescence and static light scattering measurements. Additionally, rheological measurements were conducted to study the solution viscosity and viscoelastic behavior of the mAb solutions. The so-determined analytical parameters were scored and merged in an analytical toolbox. The resulting scoring was then successfully correlated with long-term storage (40 d of incubation) experiments. Our results indicate that the sensitivity of complex rheological measurements, in combination with the applied techniques, allows reliable statements to be made with respect to the effect of solution properties, such as protein concentration, ionic strength, and pH shift, on the strength of protein-protein interaction and solution colloidal stability.     

The role of interchain disulphide bridges in the conformational stability of human immunoglobulin G1 subclass. Hydrogen-deuterium exchange studies.

The hydrogen-deuterium exchange data of human immunoglobulin G1 (IgG1) are interpreted by assuming fast fluctuations of the protein conformation, through which the peptide groups become exposed to the solvent. The probability of solvent exposure of peptide hydrogens reflects a rather loose conformation for native IgG in comparison with other globular proteins. The probability of solvent exposure is greater than 10(-3) for half of the peptide groups, which shows that the conformational transitions by which these groups are exposed to the solvent are accompanied by changes in standard free energy less than 17 kJ/mol (4 kcal/mol). In the range of pH 6.2-8.45, at 25 degrees C no gross conformational changes are reflected in the hydrogen-deuterium exchange behaviour of the native, the reduced-nonalkylated-reassociated and the reduced-S-alkylated-reassociated IgG1. No difference could be detected in the conformational stability of the native and reoxidised reassociated IgG1 proteins. The lack of inter-subunit disulphide bridges in S-alkylated-reassociated molecules results in an increased conformational motility. This destabilization of protein conformation affects about 90% of the peptide groups covered by the measurements, and corresponds to changes in standard free energy of 8 kJ/mol on the average.     

Formation of a molten globule like state in bovine serum albumin at alkaline pH

Little work has been done to understand the folding profiles of multi-domain proteins at alkaline conditions. We have found the formation of a molten globule-like state in bovine serum albumin at pH 11.2 with the help of spectroscopic techniques; like far and near ultra-violet circular dichroism, intrinsic and extrinsic fluorescence spectroscopy. Interestingly, this state has features similar to the acid-denatured state of human serum albumin at pH 2.0 reported by Muzammil et al. (Eur J Biochem 266:26–32, 1999). This state has also shown significant increase in 8-anilino-1-naphthalene-sulfonate (ANS) binding in compare to the native state. At pH 13.0, the protein seems to acquire a state very close to 6 M guanidinium hydrochloride (GuHCl) denatured one. But, reversibility study shows it can regain nearly 40% of its native secondary structure. On the contrary, tertiary contacts have disrupted irreversibly. It seems, withdrawal of electrostatic repulsion leave room for local interactions, but disrupted tertiary contacts fail to regain their original states.