Heat-induced reversible gelation of arachin: kinetics, thermodynamics and protein species involved in the process.

Arachin forms a heat-reversible gel under certain experimental conditions. The minimal gelling concentration for this system is 7.25%. Above minimal gelling concentration calculation of thermodynamic parameters for gelation of arachin revealed a constant delta Hbonding (-1220 cal.mol-1) where delta Sbonding values varied with an increase in protein concentration (ranging from -4.01 e.u. at 7.5% to -3.48 e.u. at 10.0%). The main steps involved in the gelation phenomenon include thermal denaturation of arachin, partial aggregation of heat-denatured protein molecules, setting of protein solution and maturation of the gel formed. Gel maturation process follows first order kinetics and is characterized by a large positive delta G+(+) (22,030 cal.mol-1). Determination of delta H+(+) and delta S+(+) for this process revealed that mostly delta S+(+) (-62.9 e.u.) contributes to the large positive delta G+(+), thus decreasing the overall rate of gel maturation process. This large negative delta S+(+) value probably arises from a loss of entropy of protein molecules because of their increased involvement in gel network formation. The polymer gel network seems to be primarily contributed by a part of both arachin dodecameric and hexameric species.

Structural stability of beta-lactoglobulin in the presence of kosmotropic salts. A kinetic and thermodynamic study.

The thiol group of beta-lactoglobulin reacted very sluggishly with dithio-bis-nitro-benzoic acid as compared to that of glutathione at pH 6.85. The pKapp value of the thiol group of the protein was 9.35. In the presence of 3 M urea, the thiol group reacted completely with dithio-bis-nitrobenzoic acid at pH 6.85. Heating (from 50 degrees to 80 degrees) increased the exposure of the thiol by dissociating the dimer unit. From the pseudo-first order rate constants of heat-exposure of thiol, thermodynamic activation parameters, delta G++, delta H++, and delta S++, for the heat-dissociation of beta-lactoglobulin dimer were estimated to be 23,290 cal/mol, 31,160 cal/mol, and 22.9 e.u. (at 70 degrees), respectively. Addition of kosmotropic salts, chloride, tartrate, sulfate, phosphate, and citrate (0.2 M) decreased the heat-induced exposure of the thiol group (at 70 degrees), probably by decreasing the dissociation of the dimer at pH 6.85. The relative change in free energy of activation for the dissociation of the dimer, delta(delta G++dimer), in the presence of the salts was positive, suggesting that these additives increase the stability of the dimer against heat. These salts also increased the conformational stability of beta-lactoglobulin as revealed by an increase in -delta(delta G0conf) values in their presence. Both delta(delta G++dimer) and -delta(delta G0conf) values followed the order, chloride less than tartrate less than sulfate less than phosphate less than citrate. These salts seem to manifest their structure-stabilizing effect by increasing both inter- and intramolecular hydrophobic interactions via changes in structure of water.

Effect of oxidative sulfitolysis of disulfide bonds of bovine serum albumin on its structural properties: a physiochemical study.

The effect of S-S bond cleavage of bovine serum albumin (BSA) on some of its structural properties, including solubility, viscosity, and conformation, were investigated. Cleavage of S-S bonds decreased the solubility of serum albumin and also shifted its isoelectric point to lower pH values. S-S bond cleavage resulted in changes in shape and hydrodynamic volume of the protein, increasing the specific viscosity, with cleavage of up to 14 S-S bonds, followed by a decrease with further cleavage. Both UV difference and fluorescence spectral measurements indicated that conformational flexibility increases with S-S bond cleavage. Secondary structure estimations by far UV-CD suggested a gradual decrease in alpha-helical content of the protein with progressive cleavage of its S-S bonds. However, fully S-S bond cleaved protein maintained some alpha-helical structure. Sulfitolysis of the protein also decreased its 1,8-anilino-naphthalene sulfonate-binding ability.

Enhanced thermodynamic stability of beta-lactoglobulin at low pH. A possible mechanism.

The thermodynamic stability of beta-lactoglobulin (beta-Lg) was studied at acidic and near-neutral pH values using equilibrium thermal-unfolding measurements. Transition temperature increased with a decrease in pH from 7.5 to 6.5 and 3.0 to 1.5, suggesting an increase in the net protein stability. Determination of the change in free energy of unfolding and extrapolation into the nontransition region revealed that beta-Lg increases its stability by increasing the magnitude of the change in free energy of unfolding at the temperature of maximum stability, as well as by increasing the temperature of maximum stability. The relative difference in the change in free energy of unfolding at 70 degrees C (with a reference pH of 7.5) was positive and its magnitude increased with a decrease in pH from 7.0 to 1.5 van't Hoff plots of thermal unfolding of beta-Lg at all pH values studied were non-linear and the measured changes in the enthalpy and entropy of unfolding for beta-Lg were high and positive. The relative magnitude of change of both enthalpy and entropy at 70 degrees C (compared with pH 7.5) increased with a decrease in pH up to 1.5. A possible mechanism for the increased stability of beta-Lg at low pH is discussed.

Fluorescence energy transfer studies on lima bean lectin. Distance between the subunit hydrophobic binding site and the thiol group essential for carbohydrate binding.

Measurements of the efficiency of singlet-singlet energy transfer were used to determine the distance between the hydrophobic binding site and the thiol group required for carbohydrate-binding activity of lima bean lectin. 1-Anilino-8-naphthalenesulfonate, bound to the hydrophobic binding site by noncovalent interactions, was used as the donor. Two different nonfluorescent probes were used as the acceptors: a mercurial, 2-chloromercuri-4-nitrophenol, and a maleimide, 4-dimethylaminophenylazophenyl-4'-maleimide. Acceptor was covalently attached to the thiol group at the putative carbohydrate binding site. The efficiency of energy transfer in both the 1-anilino-8-naphthalenesulfonate/2-chloromercuri-4-nitrophenol and and 1-anilino-8-naphthalenesulfonate/4-dimethylaminophenylazophenyl-4' -maleimide donor-acceptor systems indicated an apparent distance of 28 A between the two sites, assuming that the transition dipole of the donor is not correlated with respect to that of the acceptor and that each donor is quenched by a single acceptor. Using an alternate model wherein each donor is equally quenched by two acceptors on adjacent subunits, an apparent distance of 33.4 A was calculated. The fact that two donor-acceptor pairs with different Förster's critical distance parameters yielded the same distance between the sites is consistent with our assumption of uncorrelated donor-acceptor transition dipoles.