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Interaction of bovine β-lactoglobulin (BLG) with several flavor compounds (FC) (2-methylpyrazine, vanillin, 2-acetylpyridine, 2- and 3-acetylthiophene, methyl isoamyl ketone, heptanone, octanone, and nonanone) was studied by high-sensitivity differential scanning calorimetry. The denaturation temperature, enthalpy, and heat capacity increment were determined at different FC concentrations. It was found that the denaturation temperature and heat capacity increment do not depend on the FC concentration, while the denaturation enthalpy decreases linearly with the FC concentration. These thermodynamic effects disclose the preferential FC binding to the unfolded form of BLG. By the obtained calorimetric data, the free energies of FC binding vs. the FC concentrations were calculated. These dependences were shown to be linear. Their slope relates closely to the overall FC affinity for the unfolded BLG in terms of the Langmuir binding model. The overall BLG affinity for FC varies from 20 M -1 (2-methylpyrazine) up to 360 M -1 (nonanone). The maximal stoichiometry of the BLG-FC complexes was roughly estimated as a ratio of the length of the unfolded BLG to the molecular length of FC. Using these estimates, the apparent BLG-FC binding constants were determined. They are in the range of 0.3-8.0 M -1 and correlated strictly with the FC lipophilicity descriptor (logP)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Energetics of chitosan (CS) polyplexes and conformational stability of bound DNA were studied at pH 5.0 by ITC and HS-DSC, respectively. The CS-DNA binding isotherm was well approximated by the McGhee-von Hippel model suggesting the binding mechanism to be a cooperative attachment of interacting CS ligands to the DNA matrix. Melting thermograms of polyplexes revealed the transformation of different conformational forms of bound DNA in dependence on the CS/DNA weight ratio r w . At 0w <0.7 two conformational forms were observed and assigned, respectively, to the intact (free) and globular (bound) DNA. A contribution of the globular DNA to the melting enthalpy approached 100 % at the equivalent weight CS content. At higher CS contents a further stabilization of the globular DNA conformation was detected and assigned to the DNA globules in overcharged polyplexes. The polyplex dissociation was studied at pH 7.4 under conditions of zero and a physiological concentration of NaCl. Phase separation of the system was observed in both cases. DNA was almost completely immobilized in the lower phase enriched with CS. At physiological ionic strength, DNA converted from the globular to the intact form although remained to be immobilized in the CS phase., Competing Interests: Declaration of competing interest The authors do not have any conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Oligochitosan, a low molecular weight derivative of the cationic biopolymer, chitosan, currently shows a great potential of application as a biodegradable non-toxic stimuli-sensitive drug carrier. This paper aimed to elucidate the thermoresponsive potential of oligochitosan and the temperature-controlled drug binding and release to shed light on oligochitosan potential in stimuli-responsive drug delivery. Mechanisms of thermoresponsive behavior of oligochitosan induced by β-glycerophosphate (GP) were investigated using ITC, DSC, and DLS. Upon heating, the aqueous oligochitosan solution underwent a cooperative transition of the microphase separation type resulting in the formation of stable nano-sized particles. Energetics of the GP-oligochitosan interaction (evaluated by ITC) revealed a positive enthalpy of the GP binding to oligochitosan, which pointed to a notable contribution of dehydration and the related rearrangement of the polysaccharide hydration shell. Energetics of the thermal phase transition of oligochitosan was investigated by DSC upon variation of the solvent dielectric constant and GP concentration. The dependences of the transition parameters on these variables were determined and used for the analysis of the oligochitosan thermoresponsivity mechanism. The binding of ibuprofen to the thermotropic oligochitosan nanogel particles and its release from them were evaluated under near-physiological conditions. Relevantly, the oligochitosan nanoparticles surpassed some reference macromolecular adsorbers by the affinity for the drug and by the delayed release kinetics., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Most drugs besides their intended activity, express undesired side effects, including those with the engagement of cell membrane. Previously, such undesired nonspecific effects on the membrane have been shown for a number of widely used nonsteroidal anti-inflammatory drugs. In this paper, we study the mechanism of interaction between moxifloxacin (Mox), antibacterial drug of broad specificity, with lipid bilayer of the liposomes of various compositions as a model of cell membrane using a combination of spectroscopy methods, including ATR-FTIR spectroscopy, circular dichroism, UV and fluorescence spectroscopy. The fine structure of the moxifloxacin-liposome complex, localization of the drug in bilayer and the main sites of Mox interaction with lipid membrane were determined. Lipid composition of the liposome plays a key role in the interaction with moxifloxacin, drastically affecting the loading efficiency, strength and character of drug binding, lipid phase segregation and phase transition parameters. In case of anionic liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and cardiolipin (CL 2- ) the electrostatic interaction of negatively charged nitrogen in heterocycle moiety of moxifloxacin with cardiolipin phosphate groups is a crucial factor for stable complex formation. The study of moxifloxacin-liposome complex behavior at phase transition in bilayer by DSC method revealed that in DPPC/CL 2- liposomes system two microphases with different content of CL 2- coexist and Mox interacts with both of these microphases resulting in the formation of two types of complexes with different structure and phase transition temperature. This binding stabilized the gel-state of the lipid bilayer with increasing the phase transition temperature Tm up to 3-5 °C. A different situation is observed for neutral DPPC liposomes: drug interaction with bilayer results in defects formation and a fluidization effect in lipid bilayer, resulted to decrease the Tm value by 2-4 °C. Moxifloxacin is not firmly binding in the membrane of DPPC and drug releases rapidly., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Thermoresponsivity of chitosan induced by β-glycerophosphate (GP) in diluted aqueous solutions has been first studied by high-sensitivity differential scanning calorimetry. It has been found that the GP solutions of chitosan undergo a first-order phase transition upon heating. The onset of this transition coincides with the cloud point of the system. This allows one to identify the thermoresponsivity of chitosan as a macroscopic demonstration of the phase separation transition. The transition temperature, enthalpy, heat capacity increment, and width were determined as functions of GP and chitosan concentrations, and the dielectric constant of the solvent. Based on this data, we suggested that GP binds cooperatively to the chitosan matrix at low temperatures. The standard free energy of GP binding (Δ b g int = -6 ± 1 kJ mol -1 ) was estimated from the DSC data. It was shown that the Okada-Tanaka model of cooperative hydration of polymers adequately describes the thermogram of the GP induced phase transition of chitosan., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Changes in the affinity of the swollen and collapsed forms of a thermoresponsive polymer gel for targeted ligands can be directly estimated using a thermodynamic approach based on high-sensitivity differential scanning calorimetry (HS-DSC). For macromolecular ligands (proteins) bound to the gel, this method provides information on changes in their conformational stability, which is of crucial importance for the biological or pharmaceutical activity of the protein. We used HS-DSC for the study of interactions of two widely administrated drugs-gemfibrozil and ibuprofen-and two globular proteins-α-lactalbumin and BSA-with hydrogels of the cross-linked poly(methoxyethylaminophosphazene). The gel collapse resulted in a substantial increase in the gel affinity for the drugs. We obtained quantitative estimations of the affinity of the collapsed gels depending on the gel structure, pH, concentration of NaCl, and phosphate buffer (an inductor of the thermoresponsivity). The gels retained a high affinity for the drugs in the near-physiological conditions (ionic composition and pH). The binding curves of globular proteins to the gels in the swollen and collapsed states were obtained. The different proteins demonstrated the preferential binding to the swollen or collapsed state of the gels, presumably depending on the protein surface hydrophobicity. The proteins bound to the gel subchains retain their native tertiary structure and, therefore, maintain their functionality when immobilized in the polyphosphazene hydrogels.

We investigated energetics of binding of multifunctional pyranine ligands to hydrogels of the cross-linked poly(methoxyethylaminophosphazene) (PMOEAP) from data on the thermotropic volume phase transition of the gels by means of high-sensitivity differential scanning calorimetry. Dependences of the transition temperature, enthalpy, and width on the concentration of pyranines were obtained, and the excess transition free energy as a function of the pyranine concentration was calculated. We found that the affinity of the gels for the pyranine ligands increased very significantly upon the gel collapse. The intrinsic binding constants and free energies of binding of the ligands to the gels in the collapsed state were estimated from the DSC data. They revealed a significant increase in the hydrogel affinity for pyranines proportional to the number of anionic groups in the ligand structure. The affinity of the PMOEAP hydrogels for the multifunctional ligands was not affected by an increase in the cross-linking density of the gels and only slightly reduced by physiological salt concentrations.

Biodegradable hydrogels of cross-linked polymethoxyethylaminophosphazenes (PMOEAPs) of various cross-linking density and apparent subchain hydrophobicity were investigated by high-sensitivity differential scanning calorimetry and equilibrium swelling measurements. The volume phase transition of the hydrogels was found to be induced by salts of weak polybasic acids. The transition parameters were determined depending on the pH, phosphate concentration, cross-linking density, and apparent hydrophobicity of the gels. The transition enthalpy increased three times and reached 60 J g -1 at the phosphate concentrations 5-100 mM. The transition temperature decreased by 60 °C when the pH changed from 6 to 8. A decrease in the transition temperature (by ∼20 °C) was achieved due to incorporation of 9.4 mol % of some alkyl groups into the gel subchains. The classic theory of the collapse of polymer gels coupled with the data of protein science on hydration energetics for various molecular surfaces reproduces correctly thermodynamics of the collapse of PMOEAP hydrogels.

The phosphorus-containing glycerolipid based antitumor drugs (edelfosine as a prototype) are currently in clinical trials. To avoid the use of potentially harmful phosphoric reagents in the preparation of biologically active glycerolipids, and to obtain the compounds without the phosphoester bond cleavable inside the cells, we developed the synthesis of non-phosphorous glycerolipids (NPGLs) with neutral or cationic polar 'heads'. In this study, we analyzed the ability of novel NPGLs L1-L5 to interact with duplex DNA and interfere with the DNA modifying enzyme topoisomerase I (topo I). In cell-free systems, NPGLs formed highly affine complexes with DNA. Molecular docking revealed that NPGLs fitted very well into the DNA minor groove. Compounds L2 (with two long hydrophobic 'tails') and L4 (with ethylimidazolium cationic group), the most affine DNA binders, showed the best calculated energies of complex formation with DNA and topo I. The models demonstrated the binding of NPGLs to the topo I site known for interaction with conventional inhibitors. Each NPGL attenuated the topo I mediated unwinding of supercoiled DNA. Again, L2 and, to a lesser extent, L4 were the most potent topo I inhibitors. Thus, NPGLs with polar 'heads' emerge as a new class of DNA ligands and interfacial topo I antagonists.

Freeze-thaw processing of bovine serum albumin (BSA) aqueous solutions, which contain also the additives of denaturants (urea in this case) and thiol-bearing reductants [cysteine (Cys) in this case] leads to the formation of wide-pore cryogels. The properties and porous morphology of these spongy gel matrices were demonstrated to depend on the initial concentration of all precursors and on the freezing/frozen storage temperature. The optimum conditions for preparing such BSA-based cryogels were found to be as follows: [BSA] = 3-5 g dL(-1), [urea] = 0.5-2.0 mol L(-1), [Cys] = 0.01 mol L(-1), and freezing temperatures in the range of -15 to -20 °C. The size of gross pores in thus prepared cryogels is ∼50-150 μm. The spatial network of BSA-cryogels was shown to be cross-linked chemically via interchain disulfide bridges. The significant role of hydrophobic interactions in the stabilization of 3D networks of these cryogels is inferred, as well as the supposition about the relay-race sequence mechanism of the intermolecular disulfide cross-link formation is made.

Controlled drug binding and release stand among top requirements postulated for targeted drug delivery systems of the new generations. "Smart" polymers and gels are highly suitable for the controlled delivery due to their structural sensitivity to minor environmental variations. The aim of this work was to study thermoresponsive polyanionic and polycationic hydrogels of N-isopropylacrylamide copolymers with acrylic acid and N-aminopropylmethacrylamide in terms of their interaction with two widely used drugs, propranolol and ibuprofen. Binding energetics of these drugs by the gels in swollen and collapsed state was estimated by means of high-sensitivity differential scanning calorimetry. Thermodynamic parameters of the gel collapse (transition temperature, enthalpy, heat capacity increment, and width) were determined as a dependence of the drug concentrations. From these data the excess free energy of collapse was calculated as a function of drug concentration. Deconvolution of this function resulted in the evaluation of binding parameters and contributions from interactions of various types to the free energy of binding. The binding mechanism of both drugs to the swollen and collapsed gels was elucidated. Its main features are the cooperative character of the drug binding by the collapsed gel and the predominant role of the hydrophobicity of drugs in their affinity for the swollen gel.

Protease from Serratia proteamaculans (PSP) is the first known psychrophilic oligopeptidase B. The gene of S. proteamaculans 94 oligopeptidase B was cloned, sequenced and expressed in Escherichia coli. The unfolding of PSP molecule following heat treatment at 37°C by measuring fluorescence spectra was examined in parallel with the residual activity determination. The effect of PSP thermostabilization by glycerol at 37-50 °С was revealed. Calcium ions and buffer solution of low molarity cause the opposite effect - the acceleration of PSP inactivation at 37°C. The thermal stability of PSP molecule in the presence of 0-100mM CaCl2 was also investigated by means of high-sensitivity differential scanning calorimetry. The artificial reconstruction of the natural complex PSP-chaperonin from S. рroteamaculans was carried out: the stable complex (1:1) of chaperonin E. сoli GroEL with active recombinant enzyme PSP was obtained. It was shown that complex formation with chaperonin promotes PSP thermostability at 37°C., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.

The interaction of DNA with a synthetic biocompatible and biodegradable cationic polymer, poly(methylaminophosphazene) hydrochloride (PMAP·HCl), was investigated by high-sensitivity differential scanning calorimetry under conditions of strong and weak electrostatic interactions of the macroions. Thermodynamic parameters of the DNA double-helix melting were determined as a function of pH and the PMAP·HCl/DNA weight ratio. PMAP·HCL was shown to reveal two functions with respect to DNA: the polyelectrolyte function and the donor-acceptor one. The first function stabilizes the helical conformation of DNA, and the second one destabilizes it. The stabilizing effect of PMAP·HCl is of entropic origin, related to a displacement of mobile counterions from the DNA's nearest surroundings by the poly(methylaminophosphazene) charged groups. The donor-acceptor function of poly(methylaminophosphazene) dominates when its electrostatic interaction with DNA is either saturated (in the complex coacervate phase at high poly(methylaminophosphazene) concentrations) or completely suppressed (in a salt medium when the polycation carries a small charge). Under these conditions, poly(methylaminophosphazene) destabilizes DNA. It preferentially binds to the DNA coil form likely via the formation of multiple labile hydrogen bonds with the donor-acceptor groups of DNA., (© 2011 American Chemical Society)

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., (© 2011 American Chemical Society)

Interpolyelectrolyte complexes of lysozyme with thermosensitive N-isopropylacrylamide-sodium sulfonated styrene copolymers of different charge density were investigated by high-sensitivity differential scanning calorimetry (HS-DSC) at pH 4.6-7.2 and low ionic strength. A general property of the complexes for all copolymers investigated was a decrease in the conformational stability of the bound protein. This suggested the preferential binding of the unfolded protein to the polymer matrix. The isotherms of lysozyme binding to the copolymers were derived from the HS-DSC data. They indicate that the binding is irreversible and charge stoichiometric.

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.

The formation of electrostatic complexes between beta-casein and iota- and kappa-carrageenans is well-known. However, the molecular mechanism of the complexation has yet to be determined, particularly with respect to the conformational changes of the interacting macromolecules. High-sensitivity differential scanning calorimetry was used to study beta-casein/carrageenan mixtures at different pH values (3.0 to 7.5), ionic strengths (0.03 and 0.15 M), and various molar protein/polysaccharide ratios (3-400). The effects of these variables on the temperature, enthalpy, and width of the helix-coil transition of iota- and kappa-carrageenans were investigated. Neither pH nor the protein/polysaccharide ratio influenced the transition temperature of either carrageenan in the complexes. However, the transition enthalpy of both carrageenans in complexes with beta-casein decreased to zero with both decreasing pH and increasing protein/polysaccharide ratio. This may reflect an unwinding of the polysaccharide double helix induced by beta-casein, a conformational change which is fully reversible in conditions of sufficiently high ionic strength. The interaction of beta-casein with iota- and kappa-carrageenans was approximated in terms of the model of binding of large ligands to macromolecules, that provides the binding constants for these biopolymers.

This paper describes the effect of the kappa/iota-ratio on the physical properties of kappa/iota-hybrid carrageenans (synonyms: kappa-2, kappa-2, weak kappa, weak gelling kappa). To this end, a series of kappa/iota-hybrid carrageenans ranging from almost homopolymeric kappa-carrageenan (98 mol-% kappa-units) to almost homopolymeric-carrageenan (99 mol-% iota-units) have been extracted from selected species of marine red algae (Rhodophyta). The kappa/iota-ratio of these kappa/iota-hybrids was determined by NMR spectroscopy. Their rheological properties were determined by small deformation oscillatory rheology. The gel strength (storage modulus, G') of the kappa/iota-hybrids decreases with decreasing kappa-content. On the other hand, the gelation temperature of the kappa-rich kappa/iota-hybrids is independent of their composition. This allows one to control the gel strength independent of the gelation or melting temperature. The conformational order-disorder transition of the kappa/iota-hybrids was studied using optical rotation and high-sensitivity differential scanning calorimetry. High-sensitivity DSC showed that the total transition enthalpy of the kappa/iota-hybrids goes through a minimum at 60 mol-% kappa-units, whereas for the mixture of kappa- and iota-carrageenan, the total transition enthalpy is a linear function of the composition. With respect to the ordering capability, the kappa/iota-hybrid carrageenans seem to behave as random block copolymers with length sequence distributions truncated from the side of the small lengths. Intrinsic thermodynamic properties (e.g., transition temperature and enthalpy) of kappa- and iota-sequences in these copolymers are close to those of their parent homopolymers. The critical sequence length for kappa-sequences is 2-fold of that for iota-sequences.

Interpenetrated polymer networks (IPN) of poly(N-isopropylacrylamide) (PNIPA) and chitosan (two grades) were prepared by free radical polymerisation and cross-linking of PNIPA (700 mM) with bis(acrylamide) (20 mM) in chitosan solutions (1.5 wt.% in acetic acid), and subsequent immersion in glutaraldehyde solutions (0 to 0.7 vol.%) to post-cross-link the chitosan. The amount of chitosan that remained in the IPNs, after washing, was proportional to the glutaraldehyde concentration used in the post-cross-linking step; being only 50% of the theoretical when the post-cross-linking was omitted (semi-IPN). The temperature-induced phase transitions of the IPNs were followed by the changes in the swelling degree and in the thermodynamic parameters (temperature, enthalpy, heat capacity, and width of the transition), which were evaluated using high-sensitivity differential scanning calorimetry (HS-DSC). An increase in the post-cross-linking degree of chitosan caused a decrease in the enthalpy of the transition, and in the absolute value of the transition heat capacity increment (delta(t)C(p)), as well as a broadening of the heat capacity peak. This behaviour is a consequence of the subdivision, in the IPNs, of the PNIPA network in microdomains, some regions of which (surface or outer) cannot be involved in the transitions. On the other hand, changes in pH from 8 to 3 only increased the transition temperature from about 32 to 34 degrees C, despite the considerable modification that this caused in the ionisation degree of chitosan. The PNIPA/chitosan IPNs had a notably greater affinity for diclofenac than the pure PNIPA hydrogel and were able to sustain the drug release for more than 8 h in 0.9% NaCl solutions or pH 8 phosphate buffer. The IPNs with lower chitosan post-cross-linking degree showed the higher temperature-sensitive release patterns. In contrast, the temperature did not significantly affect the release rate from the most cross-linked IPNs, in which the PNIPA microdomains are smaller and the volume phase transitions are less sharper. Therefore, PNIPA microdomains play an important role in controlling the release process. In summary, the interpenetration of networks with complementary properties, such as those made with PNIPA and chitosan, make it possible to develop drug delivery systems with improved drug loading capacity (owing to chitosan) and sustained release behaviour (owing to PNIPA).

A series of iota-carrageenans containing different amounts of nu-carrageenan (0-23 monomer %) have been prepared from neutrally extracted carrageenan of Eucheuma denticulatum. nu-Carrageenan is the biochemical precursor of iota-carrageenan. The conformational order-disorder transition and rheological properties of these carrageenans were studied using optical rotation, rheometry, size exclusion chromatography coupled to multiangle laser light scattering, and high-sensitivity differential scanning calorimetry. The helix forming capacity of iota-carrageenan turns out to decrease monotonously with increasing amount of nu-units. In contrast, the rheological properties of iota-carrageenan are remarkably enhanced by the presence of a small amount of nu-units, yielding a maximum twofold increase in G' at 3% nu-units. It is concluded that the structure-forming capacity of iota-carrageenan, containing a small amount of nu-carrageenan, is significantly higher than that of pure iota-carrageenan. This phenomenon is explained in terms of the balance between the helical content and the number of cross-links between chains, taking into consideration the fact that nu-units introduce "kinks" in the chain conformation enabling neighboring chains to connect. Increasing amounts of nu-units increase the number of cross-links in the network, resulting in increased gel strength. On the other hand, a reduced length of the helical strands weakens the cross-links between the different chains and, consequently, the gel., (Copyright 2002 Wiley Periodicals, Inc. Biopolymers 65: 299-312, 2002)

The proposed biological function of beta-lactoglobulins as transporting proteins assumes a binding ability for ligands and high stability under the acidic conditions of the stomach. This work shows that the conformational stability of nonruminant porcine beta-lactoglobulin (BLG) is not consistent with this hypothesis. Thermal denaturation of porcine BLG was studied by high-sensitivity differential scanning calorimetry within the pH range 2.0-10.0. Dependences of the denaturation temperature and enthalpy on pH were obtained, which reveal a substantial decrease in both parameters in acidic and basic media. The denaturation enthalpy follows a linear dependence on the denaturation temperature. The slope of this line is 9.4 +/- 0.6 kJ.mol-1. K-1,which is close to the denaturation heat capacity increment DeltadCp = 9.6 +/- 0.5 kJ.mol-1.K-1, determined directly from the thermograms. At pH 6.25 the denaturation temperatures of porcine and bovine BLG coincide, at 83.2 degrees C. At this pH the denaturation enthalpy of porcine BLG is 300 kJ.mol-1. The denaturation transition of porcine BLG was shown to be reversible at pH 3.0 and pH 9.0. The transition profile at both pH values follows the two-state model of denaturation. Based on the pH-dependence of the transition temperature and the linear temperature dependence of the transition enthalpy, the excess free energy of denaturation, DeltadGE, of porcine BLG was calculated as a function of pH and compared with that of bovine BLG derived from previously reported data. The pH-dependence of DeltadGE is analysed in terms of the contributions of side-chain H-bonds to the protein stability. Interactions stabilizing native folds of porcine and bovine BLG are discussed.

The thermal unfolding and domain structure of Na+/K+-ATPase from pig kidney were studied by high-sensitivity differential scanning calorimetry (HS-DSC). The excess heat capacity function of Na+/K+-ATPase displays the unfolding of three cooperative domains with midpoint transition temperatures (Td) of 320.6, 327.5, 331.5 K, respectively. The domain with Td = 327.5 K was identified as corresponding to the beta subunit, while two other domains belong to the alpha subunit. The thermal unfolding of the low-temperature domain leads to large changes in the amplitude of the short-circuit current, but has no effect on the ATP hydrolysing activity. Furthermore, dithiothreitol or 2-mercaptoethanol treatment causes destruction of this domain, accompanied by significant disruption of the ion transporting function and a 25% loss of ATPase activity. The observed total unfolding enthalpy of the protein is rather low (approximately 12 J.g-1), suggesting that thermal denaturation of Na+/K+-ATPase does not lead to complete unfolding of the entire molecule. Presumably, transmembrane segments retain most of their secondary structure upon thermal denaturation. The binding of physiological ligands results in a pronounced increase in the conformational stability of both enzyme subunits.

Thermodynamic properties of aqueous solutions of iota-carrageenan as affected by KCl (0.15-1.2 M) or iota-carrageenan (0.5-6 mg/mL) content were studied by high-sensitivity differential scanning calorimetry. The polysaccharide was found to undergo two consecutive cooperative conformational transitions, which can be represented by the scheme: [H2]2<-->2H2<-->4C where C is the random coil, H2 is the double helix, and [H2]2 is the double helix dimer. The first transition follows by the "all or none" mechanism. The profile of the second transition resembles that of a second-order phase transition. The parameter sigma (of order 1), estimated for this latter transition, suggests that the stacking effect in helices of iota-carrageenan is rather small. The cooperativity of the transition is mainly defined by the loop factor. Free energies of both transitions at 273 K were calculated as a function of salt concentration. These experimental data were found to agree with Manning's theory.

We considered alpha-chymotrypsin (CT) in homogeneous water-organic media as a model system to examine the influence of enzyme chemical modification with hydrophilic and hydrophobic substances on its stability, activity and structure. Both types of modifying agents may lead to considerable stabilization of the enzyme in water-ethanol and water-DMF mixtures: (i) the range of organic cosolvent concentration at which enzyme activity (Vm) is at least 100% of its initial value is broadened and (ii) the range of organic cosolvent concentration at which the residual enzyme activity is observed is increased. We found that for both types of modification the stabilization effect can be correlated with the changes in protein surface hydrophobicity/hydrophilicity brought about by the modification. Circular dichroism studies indicated that the effects of these two types of modification on CT structure and its behavior in water-ethanol mixtures are different. Differential scanning calorimetry studies revealed that after modification two or three fractions or domains, differing in their stability, can be resolved. The least stable fractions (or domains) have properties similar to native CT.

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.

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.

Spectroscopic and thermodynamic studies of holo-alpha-lactalbumin folding show that in hydro-ethanolic media this protein structure is subjected to at least three distinct transitions induced by ethanol. As observed by spectrofluorescence and circular dichroism (CD) the first transition is only local, being associated with changes in the state of aromatic chromophores. During this transition overall tertiary structure of alpha-lactalbumin is retained. As shown by high-sensitivity differential scanning calorimetry (HS-DSC) and CD, the second transition involves breakdown of the protein tertiary structure. The final organisation of the protein into highly helical folding may be considered as the third structural transition since the unfolding and the new helix formation are time-resolved processes.

Interactions of food proteins--beta-lactoglobulin (BLG), bovine serum albumin (BSA) und ovalbumin (OA)--with vanillin and effect of thermal denaturation of the proteins on vanillin binding were studies by US-VIS spectrophotometry. This method has its origin in characteristic changes in the vanillin absorption spectrum at vanillin-protein complex formation and allows to calculate concentrations of the bound and free ligand in aqueous solutions. Thermodynamic parameters, the intrinsic association constants and the number of binding sites of the vanillin binding to the native and thermodenaturated proteins (monomers and clusters) were determined. It is shown that the vanillin affinity for the native proteins is decreased in the following order: BSA > BLG > OA. This sequence is reversed for the protein thermoclusters. The stepwise annealing allowing to derive complex protein mixtures composed of different types of the native and denatured protein mixtures composed of different types of the native and denatured protein particles was applied to thermodenaturation of BSA. The vanillin affinity for BSA is decreased in the order: native protein > denaturated monomer > denaturated clusters. Vanillin interaction with the proteins is mainly electrostatic in nature.

The change of the conformational stability and quaternary structure of the 7S globulin from french beans (phaseolin) has been investigated in the pH range 2.0-11.0 using the high-sensitivity differential scanning microcalorimetry technique. It has been established that each polypeptide chain of phaseolin consists of two thermodynamically unequivalent cooperative domains. The number and type of the side-chain hydrogen bonds which participate in the stabilization of the folded structure of each domain have been determined. The more stable domain contains six side-chain hydrogen bonds: four of the carboxylate-tyrosyl type and two of the carboxylate-histidyl type. The less stable domain contains four side-chain hydrogen bonds: two of the carboxylate-tyrosyl type and two of the carboxylate-histidyl type. All these side-chain hydrogen bonds appear to be localized within the hydrophobic interior of the domains. It has been found that the 3S form of phaseolin that is a product of the complete phaseolin dissociation at extreme pH values does not undergo any cooperative transition at heating. Consequently, this form probably has a conformation of 'molten globule' type.