Vladimir A. Sirotkin

Calorimetric and Fourier transform infrared spectroscopic study of solid proteins immersed in low water organic solvents

Calorimetric heat effects and structural rearrangements assessed by means of Fourier transform infrared (FTIR) amide I spectra were followed by immersing dry human serum albumin and bovine pancreatic alpha-chymotrypsin in low water organic solvents and in pure water at 298 K. Enthalpy changes upon immersion of the proteins in different media are in a good linear correlation with the corresponding IR absorbance changes. Based on calorimetric and FTIR data the solvents were divided into two groups. The first group includes carbon tetrachloride, benzene, nitromethane, acetonitrile, 1,4-dioxane, n-butanol, n-propanol and pyridine where no significant heat evolution and structural changes were found during protein immersion. Due to kinetic reasons no significant protein-solvent interactions are expected in such systems. The second group of solvents includes dimethyl sulfoxide, methanol, ethanol, and water. Immersion of proteins in these media results in protein swelling and involves significant exothermic heat evolution and structural changes in the protein. Dividing of different media in the two groups is in a qualitative correlation with the solvent hydrophilicity defined as partial excess molar Gibbs free energy of water at infinite dilution in a given solvent. The first group includes the solvents with hydrophilicity exceeding 2.7 kJ/mol. More hydrophilic second group solvents have this energy values less than 2.3 kJ/mol. The hydrogen bond donating ability of the solvents also assists in protein swelling. Hydrogen bonding between protein and solvent is assumed to be a main factor controlling the swelling of dry solid proteins in the studied solvents.

Hydration of Proteins: Excess Partial Volumes of Water and Proteins

High precision densitometry was applied to study the hydration of proteins. The hydration process was analyzed by the simultaneous monitoring of the excess partial volumes of water and the proteins in the entire range of water content. Five unrelated proteins (lysozyme, chymotrypsinogen A, ovalbumin, human serum albumin, and β-lactoglobulin) were used as models. The obtained data were compared with the excess partial enthalpies of water and the proteins. It was shown that the excess partial quantities are very sensitive to the changes in the state of water and proteins. At the lowest water weight fractions (w(1)), the changes of the excess functions can mainly be attributed to water addition. A transition from the glassy to the flexible state of the proteins is accompanied by significant changes in the excess partial quantities of water and the proteins. This transition appears at a water weight fraction of 0.06 when charged groups of proteins are covered. Excess partial quantities reach their fully hydrated values at w(1) > 0.5 when coverage of both polar and weakly interacting surface elements is complete. At the highest water contents, water addition has no significant effect on the excess quantities. At w(1) > 0.5, changes in the excess functions can solely be attributed to changes in the state of the proteins.

Volume changes associated with guanidine hydrochloride, temperature, and ethanol induced unfolding of lysozyme.

We studied the guanidine hydrochloride (GdnHCl)-, temperature-, and ethanol-induced unfolding of lysozyme using high-precision densitometric measurements, aiming to characterize and compare the volume changes, Δν(o), accompanying the unfolding of a protein simultaneously by different means, that is, by GdnHCl, temperature, and an organic cosolvent, EtOH. The data obtained are also compared with other means of unfolding, such as high-pressure- and dimethyl sulfoxide (DMSO)-induced denaturation. To aid in interpreting the temperature dependence of the apparent specific volume of lysozyme, we have also carried out pressure perturbation (PPC) and differential scanning calorimetry (DSC) measurements under the same solution conditions. The PPC method allows the detection of very small volume changes with high accuracy. Next to the strong temperature dependence of Δν(o), the volume changes associated with the unfolding of the protein are found to be very sensitive to the type of denaturation. The apparent specific volume decreases upon the heat- and GdnHCl-induced denaturation. The observed volume change for the GdnHCl-induced denaturation is 60% larger (i.e., more negative) than that obtained for thermal denaturation. Conversely, the apparent specific volume increases by an order of magnitude and becomes positive upon ethanol-induced denaturation, similar to the aprotic organic solvent, DMSO. Hence, depending on the type of denaturant (temperature, pressure, chemical denaturants, or cosolvents), positive and negative volume changes of unfolding are found, which can--at least in part--be attributed to the formation of different unfolded state structures (including clustering) of lysozyme. The standard Gibbs energy changes upon denaturation, ΔG(D)(o), for the various perturbation parameters are found to be similar, however, if extrapolated to zero cosolvent concentration.

Interactions of water with human serum albumin suspended in water-organic mixtures

Abstract Calorimetric enthalpy changes on suspending a partially hydrated preparation of human serum albumin (HSA) in various water-organic mixtures are discussed together with the water sorption isotherms. Experimental data indicate that suspending the HSA preparation is accompanied mainly by two processes. The first is water desorption-sorption which superficially obeys the Langmuir model. The influence of the medium on the thermodynamic parameters of water sorption can be described approximately by thermodynamic data on the solvation of water at infinite dilution. The second effect is a non-sorption process attributed tentatively to rupture of protein-protein contacts in the HSA preparation on suspending it. Depending on the nature of the solvent and its water content, such transformation of the HSA preparation can result in deviations from the Langmuir isotherm of water sorption by the suspended protein. This transformation is accompanied by the corresponding increase in the accessible surface area of the protein preparation and a significant enthalpy change. Experimental data cast doubt on the validity of the traditional opinion that the significant increase in water sorption by proteins at high water activities results from the various kinds of water-water interaction on the protein surface. It appears that the imposition of the transformation of the protein preparation on water sorption-desorption can determine both the calorimetric profile and thermodynamic data on suspending the protein preparation in various solvents.

Thermodynamics of water binding by human serum albumin suspended in acetonitrile

Abstract Heat effects resulting from the introduction of solid human serum albumin (HSA) into various water-acetonitrile mixtures were measured calorimetrically at 298 K. The amount of water bound to the suspended HSA as a function of the water content of the solvent was also determined. Introducing HSA into water-acetonitrile mixtures involves water binding according to the Langmuir isotherm with an adsorption constant K c = 1.0 ± 0.1 M −1 , enthalpy Δh = −9.0 ± 1.5 kJ mol −1 and entropy ΔS = −30 ± 6 J mol −1 K −1 . Placing HSA in the solvent has an additional heat effect of 46 ± 19 J g −1 , which is attributed to an unknown transformation of the protein preparation.

Interaction enthalpies of solid bovine pancreatic α-chymotrypsin with organic solvents: comparison with FTIR-spectroscopic data

Calorimetric heat effects and integral absorbance changes observed in the FTIR spectra were measured at immersing solid bovine pancreatic α-chymotrypsin in organic solvents and water at 298 K. Enthalpy changes upon the immersion of the enzyme in different media are in a good linear correlation with the corresponding IR-absorbance changes. Based on calorimetric and FTIR data, all the solvents were divided into two groups. The first group of solvents includes carbon tetrachloride, benzene, nitromethane, acetonitrile, 1,4-dioxane, n-butanol, n-propanol and pyridine in which no significant heat evolution and structural changes were found at the solid enzyme immersion. Second group of the solvents includes dimethyl sulfoxide, methanol, ethanol, and water. Immersion into these media, results in the solid protein swelling and involves significant exothermic heat evolution and structural changes in the protein. Dividing of different media in these two groups is in a qualitative correlation with the solvent hydrophilicity which is defined as partial excess molar Gibbs free energy of water at infinite dilution in a given solvent. The first group of solvents includes liquids with hydrophilicity exceeding 2.7 kJ/mol. The hydrophilicity of the second group solvents is <2.3 kJ/mol. Hydrogen bond donating ability of the solvents assists in the protein swelling. Hydrogen bonding between protein and solvent is assumed to be a main factor controlling the swelling of solid protein preparation in the solvents at room temperature.

Effect of chain length on interactions of aliphatic alcohols with suspended human serum albumin

Enthalpy changes on the immersion of human serum albumin (HSA) into n-butanol, n-propanol, ethanol and methanol containing different amounts of water have been measured calorimetrically at 25 degrees C. Water sorption isotherms on HSA were also determined in water-n-butanol and water-ethanol mixtures. From comparison of the calorimetric and sorption data, it was concluded that there is a significant enthalpy change on the HSA immersion into methanol and ethanol even under conditions where there is no change in the quantity of adsorbed water. No similar contribution was found in the n-butanol based suspensions. Water monolayer capacity evaluated from the Langmuir model decreases also significantly when going from ethanol to n-butanol. Considering this non water sorption contribution, values of the monolayer capacity and the shape of the experimental dependences, it was inferred that a relatively small change of the solvent molecule structure (from n-propanol to ethanol) affects strongly the interactions of the protein with the solvent.

Gibbs energies, enthalpies, and entropies of water and lysozyme at the inner edge of excess hydration

The aim of this study is to simultaneously monitor the excess partial Gibbs energies, enthalpies, and entropies of water and white egg lysozyme and demonstrate how these quantities correlate with the coverage of the protein macromolecules by water molecules. Isothermal calorimetry and water sorption measurements were applied to characterize the hydration dependencies of the excess thermodynamic functions. The excess partial quantities are found to be sensitive to changes in the water and protein states. At the lowest water weight fractions (w1), changes in the excess functions are primarily attributable to the addition of water. The transition of lysozyme from a glassy (rigid) to a flexible (elastic) state is accompanied by significant changes in the excess partial quantities. When the charged groups on the protein are covered, this transition occurs at w1 = 0.05; when the coverage of both polar and weakly interacting surface elements is complete, the excess partial quantities become hydrated at w1 > 0.5. At the highest water content, water addition has no significant effect on the excess quantities. At w1 > 0.5, changes in the excess functions solely reflect changes in the state of the protein.

Hydration of proteins: excess partial enthalpies of water and proteins.

Isothermal batch calorimetry was applied to study the hydration of proteins. The hydration process was analyzed by the simultaneous monitoring of the excess partial enthalpies of water and the proteins in the entire range of water content. Four unrelated proteins (lysozyme, chymotrypsinogen A, human serum albumin, and β-lactoglobulin) were used as models. The excess partial quantities are very sensitive to the changes in the state of water and proteins. At the lowest water weight fractions (w(1)), the changes of the excess thermochemical functions can mainly be attributed to water addition. A transition from the glassy to the flexible state of the proteins is accompanied by significant changes in the excess partial quantities of water and the proteins. This transition appears at a water weight fraction of 0.06 when charged groups of proteins are covered. Excess partial quantities reach their fully hydrated values at w(1) > 0.5 when coverage of both polar and weakly interacting surface elements is complete. At the highest water contents, water addition has no significant effect on the excess thermochemical quantities. At w(1) > 0.5, changes in the excess functions can solely be attributed to changes in the state of the proteins.

Heat effects and water sorption by human serum albumin on its suspension in water-dimethyl sulphoxide mixtures

Abstract The heat effects on suspending solid human serum albumin (HSA) in various water-dimethyl sulphoxide (DMSO) mixtures were measured calorimetrically at 298 K. The isotherm of the water sorption for HSA suspended in the water-DMSO mixtures was also measured. The recording of the calorimetric heat effects exhibits endothermic and exothermic peaks. The endothermic heat effects were estimated graphically from the calorimetric curves. These values are shown to obey the Langmuir isotherm of the water sorption. The quasi-thermodynamic constant of water adsorption (1.2 ± 0.3 M −1 ) and the monolayer formation energy (−20.1 ± 1.0 J g −1 ) were estimated from the calorimetric data with the Langmuir model. The adsorption constant (0.16 ± 0.08 M −1 ) was evaluated from fitting the water sorption isotherm by the Langmuir model also. There is a divergence between the latter constant and the adsorption constant obtained from the calorimetric data. It appears that the processes accompanying the exothermic heat evolution influence the HSAs ability to bind water. The surface area of the water monolayer was also calculated from the fitting of the water sorption isotherm. It essentially exceeds the recognised values for proteins estimated from the data for water vapour sorption. The aqueous solubility of the protein after the exposure of the HSA preparation in the water-DMSO mixtures is also essentially decreased. Hence, changes in the protein-protein interactions of a diverse nature might accompany the exothermic heat evolution on suspending HSA in water-DMSO mixtures.