Heat-and cold-setting gels of β-lactoglobulin solutions. A DSC and TEM study (original) (raw)
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Thermochimica Acta, 1994
The thermal denaturation of p-lactoglobulin solutions (8.5% protein concentration of which 5.8% was /?-lactoglobulin) has been studied by differential scanning calorimetry. For solutions of pH between 1.5 and 8, the denaturation temperature r, was maximum at pH 3.5, and the apparent enthalpy change AH,,, seemed to be constant from pH 1.5 to 6.6, and decreased significantly at pH 8.0. With addition of N-ethylmaleimide (NEM) to the P-lactoglobulin solution (pH 7) in a molar ratio of up to 1: 1, the peak temperature TP increased slightly and then remained constant, for higher molar ratio, while AH,,, decreased. With addition of dithiolthreithol (DTT), both TP and AH,,, decreased drastically. The kinetic parameters of heat denaturation were determined by the Borchardt and Daniels method as a function of pH. At pH 1.5 and 2.5, the denaturation process presented some degree of reversibility which increased with the scan rate of the first heating of the solution. The process could be described as a second-order reaction at pH < pH, and at pH 8.0. It seemed to be a first-order reaction at pH 6.0 and 6.6. In the case of irreversibility of the denaturation process, the half-life time was verified by an isothermic method. The heat-induced gelation properties, the gelation time (heat treatment at 80°C) and gelation temperature (heat treatment from 40 to 90°C at 0.1 "C min _ ') were studied with an empirical test and an Instron machine, respectively. The addition of NEM (O-20 mM) and DTT (O-32 mM) gave rise to increasing and reducing gelation time (t,) at 8O"C, respectively. The onset gelation temperature, determined from the first increase of the apparent Young's modulus, seemed to be lower than that of the denaturation temperature determined by extrapolation to O.l"C min ' of peak temperatures obtained at various scan rates. It was * Corresponding author. 0040-6031/94/$07.00 (Q 1994 -Elsevier Science B.V. All rights reserved SSoZOO40-6031(94)01878-K 388 T.X. Liu et al.lThermochimica Acta 246 (1994) 387-403 higher than that of the onset denaturation temperature, determined from the increasing edge of the heat flow measured at 5°C min-' (7S"C for pH 8 solution). The maximum apparent Young's modulus Eapp observed with increasing heat treatment displayed a higher value at pH 6.6. The experimental results are compared to published data, and they are discussed in terms of electrostatic and hydrophobic interactions and of SH/S-S interchange reactions.
International Journal of Biological Macromolecules, 1998
The effects of heat-treatment on the conformational changes of beta-lactoglobulin were monitored by differential scanning calorimetry (DSC), binding properties to 1-anilino-8-naphthalenesulphonic acid (ANS) and to 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB). The thermal transition of beta-lactoglobulin was 100% reversible on re-heating and its binding properties to the ANS fluorescent-dye and to the DTNB probe did not significantly change when the first heating was made to a temperature T < Tmax, that of the DSC maximal peak deviation of unheated solutions. When the solutions were heated to higher temperatures, the degree of reversibility of the thermal transition decreased, while the beta-lactoglobulin surface hydrophobicity increased. Furthermore, the time (tg) needed for the sol-gel state transitions was highly temperature-dependent for the solutions showing no significant reactivity with the DTNB probe, a high percentage of residual tertiary structure but a low surface hydrophobicity. For beta-lactoglobulin showing < 50% residual tertiary structure but high surface hydrophobicity, tg values were hardly temperature-dependent. The results are discussed in terms of the role of hydrophobic interactions in the aggregation process of denatured beta-lg molecules.
The Kinetics of Heat-Induced Structural Changes of< i> β-Lactoglobulin
Journal of dairy …, 2005
Heat-induced structural changes of β-lactoglobulin were studied at temperatures ranging from 67.5 to 82.5°C, and at pH 7.5. These changes were monitored by measurement of surface hydrophobicity, thiol availability, and protein solubility. Kinetic studies were conducted to quantitatively describe the contribution of hydrophobic and SH/SS interchange reactions to the thermal structural changes of β-lactoglobulin. Results indicate that β-lactoglobulin is sensitive to heat-induced interchange reactions with consequences for protein solubility. The extent of changes measured by the increase in surface hydrophobicity and the decrease in slow-reacting SH groups content could be described by a first-order fractional conversion model and were characterized by activation energy values of 233.9 ± 8.6 and 148.2 ± 6.7 kJ/mol, respectively. The break in the Arrhenius plot suggested in literature for β-lactoglobulin denaturation was confirmed in this study only for the kinetics of exposed SH groups.
Thermal unfolding of monomeric and dimeric β-lactoglobulins
European Journal of Biochemistry, 2001
The thermal stabilities of dimeric bovine b-lactoglobulin and monomeric equine b-lactoglobulin were investigated at neutral pH by means of differential scanning calorimetry, circular dichroism, tryptophan fluorescence, and by binding of an hydrophobic probe. Differential scanning calorimetry showed the presence of two structural domains with different thermal stabilities in both proteins. Thermodynamic analysis of the calorimetric signal revealed that the two domains unfold independently according to a mechanism where an equilibrium step is followed by an irreversible transition. The spectroscopic data supported this model and allowed recognition of the structural regions corresponding to the more thermally stable domain. The differences in thermal stability between the two proteins can be primarily ascribed to the properties of the less stable domain.
Heat and cold denaturation of β-lactoglobulin B
FEBS Letters, 1992
Yhc thcrmol dcnniuration of bavinc~~lastoplobulin B wus invcstlptcd by high.rcnrilivity diffcrcntial rcJinning microcalorimcuy bcrwccn pH 1-S and 3.0 in 20 mM phoqLu buffer. 'The pmczx was l'ound lo be u rcvcrsibls. two~stutc transition. Frqrcssivc uddition of gunnidinc hydrashioridc at pH 3.0 leads to the uppcarancc of u low-tcmpcralurc ulorimctric cndothcmt, corresponding to Ihe cold rcnnturation of the prokin. Ciicuhr dichroilrm experiments hnvc confirmed the low und high tcmprpturc dcnaturntion prrxcsscs. and have shown some structural diffcrcnccs brtwccn both dcnuturcd stales of &Inctoplobulin 6,
COMPARATIVE ANALYSIS OF THERMAL PROPERTIES OF TWO TYPES OF β-LACTOGLOBULIN A AND B
IASET, 2013
In this paper we have adressed to a comparative analysis of thermal denaturation properties of β-lactoglobulin types A and B. The analysis has been carried out in the absence and presence of some osmolytes and polyols with various concentrations at pH= 2.0. Our interpretation showed that the D G protein is function of sugar concentration and increased with increasing sugar concentration . On the other hand, Hm of two types lactaglobulin has an insignificant dependence on the sugar concentrations . Estimated denaturation temperatures are 351.0 K and 348.2 K for A type and B type respectively.
Calorimetric and circular dichroic studies of the thermal denaturation of β-lactoglobulin
Biophysical Chemistry, 1989
The thermal denaturatlon of &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 accompanying denaturation have been obtained. This allowed calculation of the enthalpy, entropy, and Gibbs free energy changes for thermal denaturation in the temperature range scanned, i.e., 20-100 '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 ([19]) becomes less negative, which reflects unfolding of the native protein. At the highest temperature of CD measurements, i.e., 80 o C, conformational changes are to a large extent reversible.
Changes in structure and in interactions of heat-treated bovine beta-lactoglobulin
Protein and peptide letters, 2008
Heat stress on structure and ligand binding of beta-LG has been studied by fluorescence, circular dichroism and gel electrophoresis at pH 6.5. Native PAGE gel electrophoresis shows that denaturation of beta-LG is reversible up to 75 degrees C then it becomes irreversible due to aggregation of beta-LG. Formation of aggregated beta-LG is completed at 95 degrees C. Circular dichroism results indicate that formation of aggregated beta-LG is accompanied by the scrambling of disulfide bonds (creation of new intramolecular and intermolecular disulfide bridges and rearrangement of old intramolecular disulfide bridges). Addition of ethanolic retinol causes a change in polarity of the solution and favors transformation of the beta<-->alpha structure. In the presence of retinol, the alpha-helix content of the secondary structure of heat-treated beta-LG is increased and the major portion of its secondary structure is helical. Fluorescence results show that heat-treated beta-LG at 95 degre...
Specific divalent cation-induced changes during gelation of .beta.-lactoglobulin
J Agr Food Chem, 1992
Thermally induced gelation of @-lactoglobulin sols that contained NaCl and/or CaC12 at pH 7.0 and the rheological properties of the resulting gels were investigated. Gels containing 20 mM CaCl2 were more deformable than gels containing 100 mM NaCl since a greater shear strain was required to achieve fracture. Investigating the gelation process by small-strain dynamic rheology showed that @-lactoglobulin sols containing 20 mM CaC12 gelled more rapidly and had lower gel points than similar sols containing 100 mM NaC1. Following gelation at 80 OC and cooling to 25 OC, gels containing 100 mM NaCl had greater values for G' (storage modulus) than those containing 20 mM CaC12. These cation-dependent differences in gelation and rheological properties of the gels could not be explained by cation-associated differences in structure or denaturation characteristics determined by circular dichroism measurements.
Macromolecules, 2004
The effect of the NaCl concentration (C s) on the structure factor and the turbidity of heated ovalbumin solutions was investigated using cross-correlation dynamic light scattering. The heated systems are characterized by a correlation length, which depends on protein and salt concentration, beyond which they are homogeneous. At length scales below this correlation length, the systems have a self-similar, fractal, structure. For C s g 100 mM, very turbid systems are formed with correlation lengths that show strong concentration dependence, and the correlation length rapidly rises above 1 µm at protein concentrations above 4 g/L. For Cs e 30 mM NaCl the systems remain transparent with relatively small correlation lengths (<20 nm). At 50 mM NaCl the correlation length first increases strongly with increasing protein concentration followed by a decrease above 8 g/L. At both 30 and 50 mM, an unexpected increase in the correlation length and turbidity is observed at high protein concentrations (above 40 and 30 g/L, respectively). From the present work we conclude that the structure of heated globular protein solutions and gels is generally determined by the interplay between the growth of the aggregates and the electrostatic interaction between the aggregates.