Nataša Poklar

Calorimetric and circular dichroic studies of the thermal denaturation of β-lactoglobulin

The thermal denaturation of beta-lactoglobulin in aqueous solutions at pH 5.5 and 2.0 was investigated by differential scanning calorimetry (DSC) and circular dichroic (CD) measurements. By calorimetry, the denaturation temperatures (Td), denaturation enthalpies, and specific heat capacity changes for thermal denaturation in the temperature range scanned, i.e., 20-100 degrees C. The unfolding process was found to be only partially reversible. Analysis of the far-ultraviolet CD spectra reveals that with increasing temperature the mean residue ellipticity [( theta]) becomes less negative, which reflects unfolding of the native protein. At the highest temperature of CD measurements, i.e., 80 degrees C, conformational changes are to a large extent reversible.

Studies by UV spectroscopy of thermal denaturation of β-lactoglobulin in urea and alkylurea solutions

The thermal denaturation of β-lactoglobulin in aqueous solutions at three different pHs and in aqueous solutions of urea, methyl-, N,N′-dimethyl- and ethylurea was studied by UV spectroscopy. The UV-melting curves were analyzed on the basis of two-state approximation to obtain the apparent equilibrium constant, Kapp, and the apparent standard enthalpy of transition, ΔHapp0, for protein unfolding as a function of temperature. From Kapp, calculations of ΔGapp0, as functions of temperature around transition temperature, T12, in urea and alkylurea solutions and different buffer solutions have been carried out. An increase in the observed transition temperature, T12, and the corresponding transition enthalpies, ΔHapp0, with decreasing pH or denaturant concentration indicate increased stability of protein in these conditions. However, comparison of ΔHapp0 with ΔHcal shows that the thermal transition of β-lactoglobulin in aqueous urea and alkylurea solutions is not a two-state process. It has also been observed that urea and all alkylureas cause a red shift in the absorbance spectrum of β-lactoglobulin which increases with increasing denaturant concentration and decreasing pH. A similar increase in red shift of β-lactoglobulin absorbance spectrum has also been observed with increasing temperature.

INTERACTION OF 3-ALKYLPYRIDINIUM POLYMERS FROM THE SEA SPONGE RENIERA SARAI WITH INSECT ACETYLCHOLINESTERASE

Abstract3-Alkylpyridinium polymers (poly-APS), composed of 29 or 99 N-butyl-3-butyl pyridinium units, were isolated from the marine sponge Reniera sarai. They act as potent cholinesterase inhibitors. The inhibition kinetics pattern reveals several successive phases ending in irreversible inhibition of the enzyme. To provide more information on mechanism of inhibition, interaction of poly-APS and N-butyl-3-butyl pyridinium iodide (NBPI) with soluble dimeric and monomeric insect acetylcholinesterase (AChE) was studied by using enzyme intrinsic fluorescence and light scattering, conformational probes ANS and trypsin, and SDS–PAGE. Poly-APS quenched tryptophan fluorescence emission of AChE more extensively than NBPI. Both inhibitors exhibited a pseudo-Lehrer type of quenching. Interaction of poly-APS with dimeric AChE did not induce significant changes of the enzyme conformation as assayed by using the hydrophobic probe ANS and trypsin digestion. In contrast to NBPI, titration of both monomeric and dimeric AChE with poly-APS resulted in the appearance of large complexes detected by measuring light scattering. An excess of poly-APS produced AChE precipitation as proved on SDS–PAGE. None of the effects were observed with trypsin as a control. It was concluded that AChE aggregation and precipitation rather than the enzyme conformational changes accounted for the observed irreversible component of poly-APS inhibition.

Fluorescence studies of the effect of pH, guanidine hydrochloride and urea on equinatoxin II conformation

The solvent denaturation of equinatoxin II (EqTxII) in aqueous solutions of urea, guanidine hydrochloride (Gu-HCl) and at various pH values was examined by monitoring changes in the protein intrinsic emission fluorescence spectra and in the fluorescence spectra of the added external probe ANS. It has been observed that EqTxII denaturation is reflected in a strong red shift of intrinsic fluorescence emission maxima accompanied by a simultaneous decrease in fluorescence intensity and that guanidine hydrochloride is significantly more powerful denaturant than urea or changing of pH. Comparison of intrinsic fluorescence spectra of EqTxII denatured by one of the three denaturing agents has shown that the fully denatured states of the protein in Gu-HCl and urea are similar and substantially different from those induced by changing of pH. Furthermore, according to the measurements of the ANS-fluorescence in EqTxII solutions as a function of pH the protein exists at pH values below 2.0 in an acid-denatured compact state.

Thermodynamics of denaturation of alpha-chymotrypsinogen A in aqueous urea and alkylurea solutions.

The effects of pH, urea, and alkylureas on the thermal stability ofα-chymotrypsinogen A (α-ctg A) have been investigated by differential scanning calorimetry (DSC) and UV spectroscopy. Heat capacity changes and enthalpies of transition ofα-ctg A in the presence of urea and alkylureas were measured at the transition temperature. Using these data, the corresponding Gibbs free energies, enthalpies, and entropies of denaturation at 25°C were calculated. Comparison of these values shows that at 25°C denaturation with urea is characterized by a significantly smaller enthalpy and entropy of denaturation. At all denaturant concentrations the enthalpy term slightly dominates the entropy term in the Gibbs free energy function. The most obvious effect of alkylureas was lowering of the temperature of transition, which was increasing with alkylurea concentration and the size of alkyl chain. Destabilization of the folded protein in the presence of alkylureas appears to be primarily the result of the weakening of hydrophobic interactions due to diminished solvent ordering around the protein molecules. At pH lower than 2.0,α-ctg A still exists in a very stable form, probably the acid-denatured form (A-form).

Acid- and base-induced conformational transitions of equinatoxin II.

We have investigated the acid- and base-induced conformational transitions of equinatoxin II (EqTxII), a pore-forming protein, by a combination of CD-spectroscopy, ultrasonic velocimetry, high precision densimetry, viscometry, gel electrophoresis, and hemolytic activity assays. Between pH 7 and 2, EqTxII does not exhibit any significant structural changes. Below pH 2, EqTxII undergoes a native-to-partially unfolded transition with a concomitant loss of its rigid tertiary structure and the formation of a non-native secondary structure containing additional alpha-helix. The acid-induced denatured state of EqTxII exhibits a higher intrinsic viscosity and a lower adiabatic compressibility than the native state. Above 50 degrees C, the acid-induced denatured state of EqTxII reversibly denatures to a more unfolded state as judged by the far UV CD spectrum of the protein. At alkaline pH, EqTxII undergoes two base-induced conformational transitions. The first transition occurs between pH 7 and 10 and results in a partial disruption of tertiary structure, while the secondary structure remains largely preserved. The second transition occurs between pH II and 13 and results in the complete loss of tertiary structure and the formation of a non-native, more alpha-helical secondary structure. The acid- and base-induced partially unfolded states of EqTxII form water-soluble oligomers at low salt, while at high salt (> 350 mM NaCl), the acid-induced denatured state precipitates. The hemolytic activity assay shows that the acid- and base-induced denatured states of EqTxII exhibit significantly reduced activity compared to the native state.

Denaturation behavior of α-chymotrypsinogen A in urea and alkylurea solutions: Fluorescence studies

The solvent denaturation ofα-chymotrypsinogen (α-ctg A) in aqueous solution of urea, methyl-,N,N′-dimethyl-, ethyl-, propyl- and butylurea was studied by fluorescence measurements. Data were analyzed on the assumption of a two-state approximation to obtain the apparent equilibrium constant,K∪ and the apparent Gibbs free energy of transition ΔG∪0. It has been observed that alkylsubstitution of urea significantly lowers the denaturant concentration needed to denatureα-ctg A at 25°C. Denaturation was accompanied by the red shift of emission maxima, the increase of the half-width of the fluorescence spectra, the increase of the fluorescence intensity, and the decrease of the fluorescence polarization. The differences of these fluorescence parameters observed forα-ctg A in alkylureas and urea can be ascribed to different unfolded states of the protein in different denaturant solutions. Minor differences in the extent of unfolding were confirmed by size-exclusion chromatography.

Interactions of α-chymotrypsinogen A with alkylureas

Abstract Solvation of α-chymotrypsinogen A (α-ctg A) in aqueous urea, methylurea, N,N′-dimethylurea and ethylurea was studied by density measurements. From the densities at constant molality and at constant chemical potential the preferential solvation parameters were determined. In urea and methylurea preferential solvation was observed, whereas in N,N′-dimethylurea and ethylurea at higher concentration water is preferentially bound. From preferential solvation data Gibbs free energy of transfer of α-ctg A from water to urea and alkylurea solutions were calculated. Since the enthalpies of transfer were determined previously, the entropies of transfer could also be obtained so that a complete thermodynamic description is available. An attempt is made to interpret the values of the thermodynamic quantities in terms of various interactions involved in solvation as well as to calculate the exchange constant by using the model of weak interactions. In solvation of alkylureas the hydrophobic nature of the alkyl groups is clearly reflected.

Extraction of Sugar Solution from Sugar Beet Cossettes by Electroporation and Compressive Load

Extraction of sugar solution from sugar beet cossettes has been studied at different parameters of electric pulses, with electric field intensities varying from 0.52 to 1.52 kV/cm. Sugar beet has been exposed to different number of electric pulses of different duration. Experiments where the only treatment was compressive load gave us maximum juice yield of 30.64 %, with PEF treatment added juice yield rose to a maximum of 80.32%. The whole process lasted 30 minutes. The first 5 minutes we pressed with pressure of 9.254 bars, then electric pulses were applied, followed by additional 25 minutes of pressing. To compare juice yields and specific energy consumption, we also performed experiments replacing PEF treatment with more established thermal treatment at temperatures (30, 40, 50, 60, 72) °C.