Thermodynamics and kinetics of the dismutation of superoxide anion (O2) catalyzed by superoxide dismutase (SOD) in batch reactors has been studied by on-line reaction calorimetry. The decomposition of hydrogen peroxide catalyzed by catalase is utilized as a source of oxygen and the autoxidation of pyrogallol as a source of the substrate (O2) for SOD. The molar reaction enthalpies of the SOD reaction and the pyrogallol autoxidation have been measured as! 160.1 and !218kJ/mol, respectively, at 298.15K and pH 8.0. The experimental results showed that SOD had no e!ect on the kinetic parameters or the mechanism for pyrogallol autoxidation. This autoxidation followed second-order reaction kinetics in the presence of limited oxygen ("rst order with respect to both pyrogallol and (O2), and the second-order rate constants were determined at 298.15K and pH 8.0 to be 1.25 and 1.30l/mol s in the absence and presence of SOD, respectively. A possible mechanism for the autoxidation of pyrogallol inhibited by SOD was also suggested. (2000) Elsevier Science Ltd. All rights reserved.

The oxidative refolding of reduced, denatured hen egg white lysozyme in the presence of a mixed macromolecular crowding agent containing both bovine serum albumin (BSA) and polysaccharide has been studied from a physiological point of view. When the total concentration of the mixed crowding agent is 100g/liter, in which the weight ratio of BSA to dextran 70 is 1: 9, the refolding yield of lysozyme after refolding for 4h under this condition increases 24% compared with that in the resence of BSA and 16% compared with dextran 70. A remarkable increase in the refolding yield of lysozyme by a mixed crowding agent containing BSA and Ficoll 70 is also observed. Further folding kinetics analyses show that these two mixed crowding agents accelerate the oxidative refolding of lysozyme remarkably, compared with single crowding agents. These results suggest that the stabilization effects of mixed macromolecular crowding agents are stronger than those of single olysaccharide crowding agents such as dextran 70 and Ficoll 70, whereas the excluded volume effects of mixed macromolecular crowding agents are weaker than those of single protein crowding agents such as BSA. Both the refolding yield and the rate of the oxidative refolding of lysozyme in these two mixed crowded solutions with suitable weight ratios are higher than those in single crowded solutions, indicating that mixed macromolecular crowding agents are more favorable to lysozyme folding and can be used to simulate the intracellular environments more accurately than single rowding agents do.

This paper reports a thermodynamic method for the two-substrate nzyme-catalyzed reaction by a random sequential mechanism in the presence of a chemical denaturant. This is a convenient method to produce not only the apparent molar thermodynamic constants (ΔrHm,a anΔ Ka) but also the standard thermod ynamic properties of the ction(ΔrH_111m; ΔrG_000000000m;ΔrS_m). Microcalorimetry has been used to investigate thermod ynamics of the reversible phosphoryl transfer from ATP to creatine catalyzed by rabbit muscle-type creatine kinase (MM-CK) at different concentrations of guanidine hydrochloriΔe (GuHCl). From a thermodynamic viewpoint, this enzyme-catalyzed reaction follows a rapid-equilibrium, random mechanism, i.e. the chemical steps are slower than those for binding of reagents, and there is no obligatory order of binding or release. At 298.15K, the standard enthalpy, Gibbs free energy, and entropy changes for the reaction at low concentrations of GuHCl were Δetermined by this metho to be 25.76kJ mol-1, 14.1kJ mol-1, and 38.9 J K-1mol-1, respectively, in agreement with those in the absence of GuHCl. The experimental results demonstrated the reliability of the above thermodynamic method, and indicated that inactivation of CK by low concentrations of GuHCl had no effect on the standard thermodynamic parameters for the CK-catalyzed reaction. A novel method for the determination of creatine kinase activity, the microcalorimetric assay for CK, was also proposed in this paper. The experimental results showeΔ that GuHCl had a noticeable inØuence on the activity of CK.

Xanthine oxidase (XO) and copper, zinc superoxide dismutase (Cu, Zn-SOD) are function-related proteins in vivo. Thermodynamics of the interaction of bovine milk XO with bovine erythrocyte Cu, Zn-SOD has been studied using isothermal titration calorimetry (ITC) and fluorescence spectroscopy. The binding of XO to Cu, Zn-SOD is driven by a large favorable enthalpy decrease with a large unfavorable entropy reduction, and shows strong entropy-enthalpy compensation and weak temperature-dependence of Gibbs free energy change. An unexpected, large positive molar heat capacity change of the binding, 3.02kJ mol−1K−1, at all temperatures examined suggests that either hydrogen bond or long-range electrostatic interaction is a major force for the binding. XO quenches the intrinsic fluorescence of Cu, Zn-SOD and causes a small red shift in the fluorescence emission maximum of the protein. A small salt concentration dependence of the binding affinity measured by fluorescence spectroscopy and a large unfavorable change in entropy for the binding measured by ITC suggest that long-range lectrostatic forces do not play an important role in the binding. These results indicate that XO binds to Cu, Zn-SOD with high affinity and that hydrogen bond is a major force for the binding.

The unfolding of rabbit muscle-type creatine kinase (MM-CK) induced by guanidine hydrochloride (Gullcl) has been studied by isothermal microcalorimetry, It has been found that the decrease in the activity Of MM-CK in dilute GuHCl solution is due to a slight perturbation of the active site conformation by dilute GuHCl, but not by a reversible inhibition by GuHCl binding at the active site or dissociation of the dimeric protein, The inactivation Of MM-CK precedes the overall conformation change of this enzyme during denaturation by GuHCl, providing a thermodynamic evidence for the proposition that the active site of an enzyme is situated in a limited region more flexible than the enzyme molecule as a whole, The intrinsic enthalpy, Gibbs free energy, and entropy changes for formation of an intermediate state Of MM-CK in the presence of moderate GuHCl concen ations at 25.OO℃ have been determined to be 260. 12.2kJ mol-1. and 830J mol-1K-1. respectively. Further unfolding Of MM-CK is observed when GuHCl concentration is higher than 3.00mol dm-3. and the protein is almost fully unfolded at 5.OOmol dm-3 GuHCl reached. The intrinsic enthalpy. Gibbs free energy. and entropy changes for formation of the unfolded state of MM-CK at 25.OO℃ have been measured as 8600. 23.0kJ mol-1. and 29kJ mol-1 K-1. respectively. The experimental results indicate that the unfolding Of MM-CK by GuHCl exhibits remarkable enthalpy-entropy compensation and the water reorganization is involved in the unfolding reaction.