Tatiana V. Burova

Effects of polysaccharides upon the functional properties of 11 S globulin of broad beans

Abstract The effect of polysaccharides upon the conformational stability, emulsifying properties and thermal gelation ability of the 11 S globulin (legumin) from broad beans under conditions of both protein-polysaccharide incompatibility and complex formation has been investigated. Under conditions of incompatibility (pH 7·6 for carboxyl-containing and neutral polysaccharides) the conformational stability of the legumin is not changed by the presence of the polysaccharides. At the same time a significant decrease in the emulsifying threshold and an increase in the stability of oil-in-water emulsions, stabilized by the protein, as well as some decrease in the gelation threshold in the presence of polysaccharides have been observed. Under conditions of legumin-polysaccharide complex formation (pH 4·2–6·0, low ionic strength for carboxyl- and sulphate-containing polysaccharides) a significant decrease in the temperature and enthalpy of legumin denaturation as well as a decrease in the minimum biopolymer concentration required for emulsions formation and an increase in emulsion stability have been observed. As a rule, legumin-polysaccharide complexes were poorly soluble even at a substantial excess of polysaccharide. Their solubility which was independent of the method of mixing was a limiting factor for preparing gels and stable foams on using these complexes.

Cloning, sequencing, expression, and characterization of thermostability of oligopeptidase B from Serratia proteamaculans, a novel psychrophilic protease

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.

Binding Energetics of Lysozyme to Copolymers of N-Isopropylacrylamide with Sodium Sulfonated Styrene

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.

High-sensitivity modulation differential scanning calorimetry of protein denaturation

Method of high-sensitivity modulation differential scanning calorimetry was applied for investigation of the kinetically controlled irreversible thermal denaturation of the trypsin inhibitor from soybeans (Kunitz inhibitor, KI) in diluted solution. The measurements were carried out with a temperature-modulation capillary nanocalorimeter designed and produced by the Institute of Biological Instrumentation of the RAS (Pushchino, Russia). An algorithm of the experimental data processing and corresponding software were developed. It was shown that the modulation nanocalorimetry allows one to obtain in one experiment the temperature dependence of the rate constant for irreversible protein denaturation. The temperature dependence of the rate constant and the activation energy of the irreversible denaturation of Kunitz inhibitor were determined. The obtained value of the activation energy (Eaxa0=xa0206xa0±xa06xa0kJxa0mol−1) agrees with independent estimates of this kinetic parameter.

Salt-Induced Thermoresponsivity of Cross-Linked Polymethoxyethylaminophosphazene Hydrogels: Energetics of the Volume Phase Transition

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.