Thermodynamic Characterization of Interactions of Native Bovine Serum Albumin with Highly Excluded (Glycine Betaine) and Moderately Accumulated (Urea) Solutes by a Novel Application of Vapor Pressure Osmometry

We used static and dynamic light scattering for comparing the mass (MW) and hydrodynamic radius (R(h)) of several hemoglobin systems, namely human hemoglobin, bovine hemoglobin, human hemoglobin cross-linked with a sebacyl residue, and bovine hemoglobin cross-linked with an adipoyl residue. We measured the MW and R(h) of these systems in 0.1M phosphate buffer at pH 7.0 in the absence and in the presence of either betaine or glycerol up to 1.7 molal concentrations. The 90 degrees scattering was measured with a photon counting machine equipped with a diode laser at 783 nm. The Rayleigh ratio [R(theta)] of the instrument was estimated using R(theta) = 7.19E-6 cm(-1) for toluene at 783 nm. The refractive index increment of hemoglobin solutions was measured using a laser beam at 750 nm. We estimated a value dn/dc = 0.210 cm3/g in the absence and dn/dc = 0.170 in the presence of 1.7 molal osmolites. For all systems both in liganded and unliganded form, the static light scattering data showed a 16% mass increase with increasing concentration of osmolites. The hydrodynamic radii of all investigated systems in the presence and absence of osmolites were close to 3.17 nm. Assuming a partial specific volume nu = 0.739 for hemoglobin, and using spherical geometry, the estimated average hydration volume of hemoglobin was 32.6 L/mole in the absence of osmolites. It decreased to 23.5 L/mole in the presence of 1.7 molal osmolites. Assuming that the density of water in the hydration volume is D = 1.0 g/cm3, the hydration of Hb was 0.51 gH2O/gHb, with a surface density of 0.20 molH2O/A2. The hydration decreased to 0.33 gH2O/gHb and 0.14 molH2O/A2 in the presence of 1.7 molal osmolites. The decreased hydration was compensated by the increased mass (i.e., decreased surface area per unit volume) so that the thickness of the water shell around these proteins remained close to a single layer of water molecules. These findings indicate that the combination of static and dynamic light scattering offer unique means for investigating the relevance of water activity on the structure and function of biological macromolecules. In the case of hemoglobin, the data suggest that the decreased oxygen affinity in the presence of osmolites reported by Colombo et al. (M. F. Colombo, D. C. Rau, and V. A. Parsegian Science, 1992, Vol. 256, pp. 655-659), as due to ligand linked water binding on hemoglobin surface, is part of a complex phenomenon involving the hydration shell of hemoglobin and the formation of low affinity supertetrameric molecules.

show abstract

“…5 This confirms the reliability of our assumptions on the quasi sphericity of the systems, and of the new approach.…”

Section: The Spherical Geometry Approachsupporting

confidence: 75%

Static and dynamic light scattering approach to the hydration of hemoglobin and its supertetramers in the presence of osmolites

et al. 2001

View full text Add to dashboard Cite

show abstract

“…The preferential exclusion of these solutes from the surface of folded bovine serum albumin, a representative globular protein, is ranked in the order GB Ͼ proline Ͼ TMAO. Trehalose and K ϩ glutamate (which accumulate in bacteria not offered an exogenous solute) are less strongly excluded than TMAO (69,70). The impacts of GB, proline, and TMAO on protein stability (the transition between folded and denatured states) have also been extensively documented (50,51), usually by measuring each solute's ability to protect proteins from urea-induced or thermal denaturation.…”

Section: Discussionmentioning

confidence: 99%

Analysis of Strains Lacking Known Osmolyte Accumulation Mechanisms Reveals Contributions of Osmolytes and Transporters to Protection against Abiotic Stress

Murdock

Burke

Coumoundouros

et al. 2014

Appl Environ Microbiol

View full text Add to dashboard Cite

Osmolyte accumulation and release can protect cells from abiotic stresses. In Escherichia coli, known mechanisms mediate osmotic stress-induced accumulation of K ؉ glutamate, trehalose, or zwitterions like glycine betaine. Previous observations suggested that additional osmolyte accumulation mechanisms (OAMs) exist and their impacts may be abiotic stress specific. Derivatives of the uropathogenic strain CFT073 and the laboratory strain MG1655 lacking known OAMs were created. CFT073 grew without osmoprotectants in minimal medium with up to 0.9 M NaCl. CFT073 and its OAM-deficient derivative grew equally well in high-and low-osmolality urine pools. Urine-grown bacteria did not accumulate large amounts of known or novel osmolytes. Thus, CFT073 showed unusual osmotolerance and did not require osmolyte accumulation to grow in urine. Yeast extract and brain heart infusion stimulated growth of the OAM-deficient MG1655 derivative at high salinity. Neither known nor putative osmoprotectants did so. Glutamate and glutamine accumulated after growth with either organic mixture, and no novel osmolytes were detected. MG1655 derivatives retaining individual OAMs were created. Their abilities to mediate osmoprotection were compared at 15°C, 37°C without or with urea, and 42°C. Stress protection was not OAM specific, and variations in osmoprotectant effectiveness were similar under all conditions. Glycine betaine and dimethylsulfoniopropionate (DMSP) were the most effective. Trimethylamine-N-oxide (TMAO) was a weak osmoprotectant and a particularly effective urea protectant. The effectiveness of glycine betaine, TMAO, and proline as osmoprotectants correlated with their preferential exclusion from protein surfaces, not with their propensity to prevent protein denaturation. Thus, their effectiveness as stress protectants correlated with their ability to rehydrate the cytoplasm.

show abstract

“…Differential scanning calorimetry (DSC (3, 4, 42)), hydrogen exchange (43)(44)(45), folding kinetic studies (46), vapor phase osmometry (47,48), densitometry (2,8), and differential refractometry (8) have been used to dissect out the mechanism of osmolyte stabilization of proteins. Initial models of osmolyte stabilization focused on exclusion of osmolyte from the protein surface (2,49) and preferential hydration of the native state (2).…”

Section: Discussionmentioning

confidence: 99%

Osmolytes Stabilize Ribonuclease S by Stabilizing Its Fragments S Protein and S Peptide to Compact Folding-competent States

Ratnaparkhi¹,

Varadarajan²

2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

Osmolytes stabilize proteins to thermal and chemical denaturation. We have studied the effects of the osmolytes sarcosine, betaine, trimethylamine-N-oxide, and taurine on the structure and stability of the protein⅐peptide complex RNase S using x-ray crystallography and titration calorimetry, respectively. The largest degree of stabilization is achieved with 6 M sarcosine, which increases the denaturation temperatures of RNase S and S pro by 24.6 and 17.4°C, respectively, at pH 5 and protects both proteins against tryptic cleavage. Four crystal structures of RNase S in the presence of different osmolytes do not offer any evidence for osmolyte binding to the folded state of the protein or any perturbation in the water structure surrounding the protein. The degree of stabilization in 6 M sarcosine increases with temperature, ranging from ؊0.52 kcal mol ؊1 at 20°C to ؊5.4 kcal mol ؊1 at 60°C. The data support the thesis that osmolytes that stabilize proteins, do so by perturbing unfolded states, which change conformation to a compact, folding competent state in the presence of osmolyte. The increased stabilization thus results from a decrease in conformational entropy of the unfolded state.Osmolytes are molecules used in nature to protect organisms against stresses of high osmotic pressure. These compounds have also been found to stabilize the native state of proteins relative to the unfolded state. The mechanism of this stabilization is not completely understood (1-4), although it is believed to result primarily from an unfavorable free energy of interaction between the osmolyte and the unfolded state of the protein (5, 6). Proteins retain activity in the presence of osmolytes suggesting that native state structure and dynamics are not greatly perturbed. However, there is little high resolution structural information available for proteins in the presence of osmolytes. The main classes of osmolytes are sugars, methyl ammonium derivatives, polyhydric alcohols, and amino acids and their derivatives (1). Molar concentrations of all the above classes of molecules have been shown to stabilize proteins. Many organisms accumulate osmolytes under conditions of water stress, such as high salinity, desiccation or freezing. In vivo, amino acid derivatives also counteract the accumulation of urea, which is capable of denaturing proteins. Marine cartilaginous fishes use, as osmolytes, a combination of urea and methylamines, i.e. a denaturant and a stabilizer, in a 2:1 ratio (1,7,8). Stability studies on RNase T1, RNase A, and other proteins have shown that, if the 2:1 ratio of urea:methylamine is maintained, the methylamine is able to counteract the destabilizing effect of the denaturant (1,7,8).In an effort to further our understanding of the basis of osmolyte stabilization of proteins, we have studied the stabilization of the fragment complementation system ribonuclease S (RNase S) 1 in four structurally similar osmolytes, namely sarcosine, betaine, trimethylamine-N-oxide (TMAO), and taurine. RNase S is a complex of two fragm...

show abstract

Thermodynamic Characterization of Interactions of Native Bovine Serum Albumin with Highly Excluded (Glycine Betaine) and Moderately Accumulated (Urea) Solutes by a Novel Application of Vapor Pressure Osmometry

Cited by 84 publications

References 36 publications

Static and dynamic light scattering approach to the hydration of hemoglobin and its supertetramers in the presence of osmolites

Static and dynamic light scattering approach to the hydration of hemoglobin and its supertetramers in the presence of osmolites

Analysis of Strains Lacking Known Osmolyte Accumulation Mechanisms Reveals Contributions of Osmolytes and Transporters to Protection against Abiotic Stress

Osmolytes Stabilize Ribonuclease S by Stabilizing Its Fragments S Protein and S Peptide to Compact Folding-competent States

Contact Info

Product

Resources

About