Valerij Ya. Grinberg

Thiol-induced oligomerization of α-lactalbumin at high pressure

Denaturation and aggregation ofα-lactalbumin at high pressure (up to 10 kbar, 1000 MPa) were studied by means of circular dichroism, gel-permeation chromatography, sodium dodecyl sulfate and gel electrophoresis. It was found that the unfolding ofα-lactalbumin at high pressure is reversible even in basic pH and at a protein concentration as large as 10%. In these conditions only a negligible fraction of the protein is denatured irreversibly and aggregates. The rate of aggregation ofα-lactalbumin at high pressure increases significantly in the presence of low-molecular reducing agents such as cysteine, 2-mercaptoethanol, and dithiothreitol. Maximal yield ofα-lactalbumin oligomerization (over 90%) was achieved in the presence of cysteine at the molar cysteine/protein ratioq=2 and atpH 8.5. Apparent molecular weight of the obtained oligomers was over 500 kDa. It was shown that the size distribution of oligomers can be modulated by varyingpH and reducing agent. The size distribution shifts in the direction of very large, poorly soluble particles whenpH decreases. Maximal content of the insoluble fraction (about 30%) can be reached at pH 5.5 when cysteine (q=2) is used as reducing agent. The oligomers ofα-lactalbumin are stabilized mainly by nonnative interchain disulfide bridges. Circular dichroism measurements point to an additional mechanism of cohesion of polypeptide chains in the oligomers, which is formation of intermolecularβ-sheets.

Interpretation of DSC data on protein denaturation complicated by kinetic and irreversible effects

Thermal denaturation of Kunitz soybean trypsin inhibitor (KTI) and ribulose-1,5-biphosphate carboxylase (RBPC) from tobacco leafs was studied by the method of high-sensitivity differential scanning calorimetry (HS-DSC). The dependence of the denaturation temperature on the heating rate reveals in the case of both proteins a non-equilibrium character of the denaturation transition in applied conditions. Developed kinetic approach allows the determination of an equilibrium transition temperature as well as the rate constants of denaturation and renaturation from the complex data of HS-DSC. This method was applied to the analysis of the pH-induced change of the conformational stability of KTI within pH range from 2.0 to 11.0. It allowed the determination of the pH dependencies: of the excess free energy of denaturation, of the activation enthalpy and entropy of denaturation as well as of the denaturation rate constant. Conclusions have been made suggesting the contribution of side-chain hydrogen bonds in the stabilisation of the native and activated states of KTI.

Calorimetric evidence for a native-like conformation of hen egg-white lysozyme dissolved in glycerol.

Hen egg-white lysozyme, lyophilized from aqueous solutions of different pH (from pH 2.5 to 10.0) and then dissolved in water and in anhydrous glycerol, has been studied by high-sensitivity differential scanning microcalorimetry over the temperature range from 10 to 150 degrees C. All lysozyme samples exhibit a cooperative conformational transition in both solvents occurring between 10 and 100 degrees C. The transition temperatures in glycerol are similar to those in water at the corresponding pHs. The transition enthalpies in glycerol are substantially lower than in water but follow similar pH dependences. The transition heat capacity increment in glycerol does not depend on the pH and is 1.25+/-0.31 kJ mol(-1) K(-1), which is less than one fifth of that in water (6. 72+/-0.23 kJ mol(-1) K(-1)). The thermal transition in glycerol is reversible and equilibrium, as demonstrated for the pH 8.0 sample, and follows the classical two-state mechanism. In contrast to lysozyme in water, the protein dissolved in glycerol undergoes an additional, irreversible cooperative transition with a marginal endothermic heat effect at temperatures of 120-130 degrees C. The transition temperature of this second transition increases with the heating rate which is characteristic of kinetically controlled processes. Thermodynamic analysis of the calorimetric data reveals that the stability of the folded conformation of lysozyme in glycerol is similar to that in water at 20-80 degrees C but exceeds it at lower and higher temperatures. It is hypothesized that the thermal unfolding in glycerol follows the scheme: N ifho-MG-->U, where N is a native-like conformation, ho-MG is a highly ordered molten globule state, and U is the unfolded state of the protein.

Effect of polysaccharides on the stability and renaturation of soybean trypsin (Kunitz) inhibitor

Thermal denaturation and renaturation of soybean trypsin (Kunitz) inhibitor (STI) were studied by high-sensitivity differential scanning calorimetry in the presence of polysaccharides (dextran, and κ-carrageenans, gum arabic, pectins and dextran sulfate). This study was carried out under conditions of both thermodynamic incompatibility and complex formation of STI and polysaccharides. The presence of polysaccharides did neither influence the denaturation temperature nor the denaturation enthalpy of STI under conditions of their incompatibility with the protein. No polysaccharide (except gum arabic) affected the ability of STI to renature and recover its inhibitory activity after thermal denaturation. At acidic pH values, the protein was shown to form electrostatic complexes with pectins and dextran sulfate. Substantial destabilisation of STI bound to dextran sulfate was observed. In the case of STI/pectin complexes, either a decrease or increase in the stability of STI was observed depending on the complex composition and esterification degree of pectin. The mechanism behind the changes in stability of STI bound to the polysaccharide matrix is discussed. Thermal denaturation of STI in complexes with dextran sulfate and pectin was completely irreversible. This observation indicates a possibility of suppressing antinutritional activities of trypsin inhibitors in soy products.

Ethanol‐induced conformational transitions in holo‐α‐lactalbumin: Spectral and calorimetric studies

Conformational transitions of holo-alpha-lactalbumin in a hydro-ethanolic cosolvent system was studied by spectrofluorescence, CD in near- and far-uv regions, and high-sensitivity differential scanning calorimetry. Experimental results allow us to propose that in isothermal conditions alpha-lactalbumin undergoes a number of conformational transitions with increasing ethanol concentration: N<=>I<=>D<=>H. The existence of I-state was deduced from spectrofluorometric and near-uv CD data. In this state the aromatic chromophores of the amino acid side chains are more accessible to the solvent displaying higher local mobility. The H-state was detected from far-uv CD spectra as a state corresponding to the content of alpha-helices higher than originally found in native protein. However, calorimetric measurements provide data revealing only the two-state mechanism of alpha-lactalbumin unfolding in both water and in aqueous ethanol solutions. This indicates that the energy levels of N- and I-states as well as of D- and H-states are similar. Thermodynamics of the unfolding of alpha-lactalbumin in hydroethanolic solutions was analyzed with the help of the linear model of solvent denaturation. Unfolding increments of enthalpy, entropy, and Gibbs energy of transfer of the protein from a reference aqueous solution to hydro-ethanolic solutions of different concentrations were determined from the calorimetric data. They are linear functions of molar ethanol fraction. The slope of the unfolding increment of Gibbs energy of transfer was calculated from data on transfer of amino acid residues taking into account the average solvent accessibility of amino acid residues in the native structure of small globular proteins, using the additive group contribution method.

Thermodynamic and kinetic stability of penicillin acylase from Escherichia coli

Thermal denaturation of penicillin acylase (PA) from Escherichia coli has been studied by high-sensitivity differential scanning calorimetry as a function of heating rate, pH and urea concentration. It is shown to be irreversible and kinetically controlled. Upon decrease in the heating rate from 2 to 0.1 K min(-1) the denaturation temperature of PA at pH 6.0 decreases by about 6 degrees C, while the denaturation enthalpy does not change notably giving an average value of 31.6+/-2.1 J g(-1). The denaturation temperature of PA reaches a maximum value of 64.5 degrees C at pH 6.0 and decreases by about of 15 degrees C at pH 3.0 and 9.5. The pH induced changes in the denaturation enthalpy follow changes in the denaturation temperature. Increasing the urea concentration causes a decrease in both denaturation temperature and enthalpy of PA, where denaturation temperature obeys a linear relation. The heat capacity increment of PA is not sensitive to the heating rate, nor to pH, and neither to urea. Its average value is of 0.58+/-0.02 J g(-1) K(-1). The denaturation transition of PA is approximated by the Lumry-Eyring model. The first stage of the process is assumed to be a reversible unfolding of the alpha-subunit. It activates the second stage involving dissociation of two subunits and subsequent denaturation of the beta-subunit. This stage is irreversible and kinetically controlled. Using this model the temperature, enthalpy and free energy of unfolding of the alpha-subunit, and a rate constant of the irreversible stage are determined as a function of pH and urea concentration. Structural features of the folded and unfolded conformation of the alpha-subunit as well as of the transition state of the PA denaturation in aqueous and urea solutions are discussed.

Ternary Interpolyelectrolyte Complexes Insulin-Poly(methylaminophosphazene)-Dextran Sulfate for Oral Delivery of Insulin

Ternary interpolyelectrolyte complexes of insulin with biodegradable synthetic cationic polymer, poly(methylaminophosphazene) hydrochloride (PMAP), and dextran sulfate (DS) were investigated by means of turbidimetry, dynamic light scattering, phase analysis, and high-sensitivity differential scanning calorimetry. Formation of ternary insoluble stoichiometric Insulin-PMAP-DS complexes was detected under conditions imitating the human gastric environment (pH 2, 0.15 M NaCl). A complete immobilization of insulin in the complexes was observed in a wide range of the reaction mixture compositions. The ternary complexes were shown to dissolve and dissociate under conditions imitating the human intestinal environment (pH 8.3, 0.15 M NaCl). The products of the complex dissociation were free insulin and soluble binary Insulin-PMAP complexes. The conformational stability of insulin in the soluble complexes of various compositions was investigated by high-sensitivity differential scanning calorimetry. The dependence of the excess denaturation free energy of insulin in these complexes on the PMAP content was obtained. The binding constants of the folded and unfolded forms of insulin to the PMAP polycation were estimated. Proteolysis of insulin involved in the insoluble ternary complexes by pepsin was investigated under physiological conditions. It was found that the complexes ensure an almost 100% protection of insulin against proteolytic degradation. The obtained results provide a perspective basis for development of oral insulin preparations.

Reducer driven baric denaturation and oligomerisation of whey proteins

The influence of high pressure on alpha-lactalbumin (ALA)/beta-lactoglobulin (BLG) mixtures of various compositions was studied at pH 8.5 by gel-permeation chromatography and sodium dodecyl sulphate (SDS) gel electrophoresis without 2-mercaptoethanol. High-molecular protein disulfide oligomers formed after denaturation by the pressure of 10 kbar (1000 MPa) if the weight fraction of BLG (W(BLG)(0)) in the protein mixture exceeded 0.2. The maximum yield of these oligomers of order 80-85% is observed at W(BLG)(0)>/=0.4. Conversions of both proteins in the oligomers are roughly the same. The estimates of the oligomerisation yield obtained by the gel-permeation chromatography and SDS gel electrophoresis agree well. This indicates that the formation of intermolecular disulfide bonds is necessary for the oligomerisation. Thus, the oligomerisation of pressure denatured ALA and BLG is driven by the thiol<-->disulfide exchange rendered possible by the vigorous baric denaturation and the exposure of the free thiol group of BLG, which acts as an initiator of disulfide bridges scrambling.

Ionic and Polyampholyte N-Isopropylacrylamide-Based Hydrogels Prepared in the Presence of Imprinting Ligands: Stimuli-Responsiveness and Adsorption/Release Properties

The conformation of the imprinted pockets in stimulus-responsive networks can be notably altered when the stimulus causes a volume phase transition. Such a tunable affinity for the template molecule finds interesting applications in the biomedical and drug delivery fields. Nevertheless, the effect that the binding of the template causes on the stimuli-responsiveness of the network has barely been evaluated. In this work, the effect of two ionic drugs used as templates, namely propranolol hydrochloride and ibuprofen sodium, on the responsiveness of N-isopropylacrylamide-based hydrogels copolymerized with acrylic acid (AAc) and N-(3-aminopropyl) methacrylamide (APMA) and on their ability to rebind and to control the release of the template was evaluated. The degree of swelling and, in some cases, energetics (HS-DSC) of the transitions were monitored as a function of temperature, pH, and concentration of drug. Marked decrease in the transition temperature of the hydrogels, accompanied by notable changes in the transition width, was observed in physiological NaCl solutions and after the binding of the drug molecules, which reveals relevant changes in the domain structure of the hydrogels as the charged groups are shielded. The ability of the hydrogels to rebind propranolol or ibuprofen was quantified at both 4 and 37 °C and at two different drug concentrations, in the range of those that cause major changes in the network structure. Noticeable differences between hydrogels bearing AAc or APMA and between imprinted and non-imprinted networks were also observed during the release tests in NaCl solutions of various concentrations. Overall, the results obtained evidence the remarkable effect of the template molecules on the responsiveness of intelligent imprinted hydrogels.

Conformational Energetics of Interpolyelectrolyte Complexation between ι-Carrageenan and Poly(methylaminophosphazene) Measured by High-Sensitivity Differential Scanning Calorimetry

The interaction of poly(methylaminophosphazene) hydrochloride (PMAP·HCl) of varying degrees of ionization (f) with the potassium salt of ι-carrageenan was studied by high-sensitivity differential scanning calorimetry at a KCl concentration of 0.15 M, which is included for the purpose of stabilizing the helix conformation of the polysaccharide up to 55 °C. The conditions of strong (pH 3.8, I = 0.15), moderate (pH 7.4, I = 0.15), and weak (pH 7.4, I = 0.25) electrostatic interactions of the polyelectrolytes were considered. The thermodynamic parameters of the helix-coil transition of ι-carrageenan were determined as a function of the polycation/polyanion ratio. We show that the interpolyelectrolyte reaction between PMAP·HCl and ι-carrageenan results in a complete unfolding of the polysaccharide helix under conditions of strong electrostatic interaction and increases its stability under conditions of medium and weak electrostatic interactions. The formation of stoichiometric PMAP-carrageenan interpolyelectrolyte complexes proceeded via a cooperative mechanism at pH 3.8 (f = 0.5) and pH 7.4 (f = 0.2) at an ionic strength of 0.15. In contrast, the complexation at pH 7.4 and an ionic strength of 0.25 could be considered to be a consecutive competitive binding of charged units of poly(methylaminophosphazene) to the oppositely charged polysaccharide matrix in the helix or coil conformation. Binding constants of the polycation to the helix and coil forms of ι-carrageenan were estimated. They revealed a preferential binding of the polycation to the helix form of the polysaccharide.