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.

Microcalorimetry was used to measure the change in enthalpy for the scission of calf thymus DNA (ct-DNA) induced by adriamycin (ADM) in the presence of ferric ions, Vitamin C, and oxygen. At 298.15K and pH 7.4, the overall molar reaction enthalpy for this cleavage was-147.1kJ/mol, noticeably higher than that by the mixture of Fe3á, Vitamin C, and O2. Under the same conditions, the enthalpy change for the damage of ct-DNA by the mixture of adriamycin, ferrous ions, and oxygen, however, was nearly zero, indicating that this mixture can not induce any detectable degradation of DNA. These esults suggest that both the activated adriamycin and hydroxyl radical attack DNA strands during the cleavage. A possible mechanism for the cleavage of DNA induced by adriamycin is proposed based on the calorimetric measurements. A novel thermodynamic model for the interactions of DNA with small molecules is also suggested. This is a convenient method tocalculate both the binding constant (Kb) and the standard thermodynamic parameters (ΔbH0m, ΔbG0m, and ΔbS0m) for the binding of adriamycin-Fe3á complex to ct-DNA by the calorimetric data. This nucleotide binding reaction is driven by a favorable enthalpy change, with a large unfavorable entropy change. This result indicates that the binding results in structural changes accompanied by an increase in the order of the whole system, implying that an intercalation mode is involved in adriamycinmediated breakage of DNA.

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.

Aluminum is a known neurotoxic agent and its neurotoxic effects may be due to its binding to DNA. However, the mechanism for the interaction of aluminum ions with DNA is not well understood. Here, we report the application of isothermal titration calorimetry (ITC), fluorescence spectroscopy, and UV spectroscopy to investigate the thermodynamics of the binding of aluminum ions to calf thymus DNA (CT DNA) under various pH and temperature conditions. The binding reaction is driven entirely by a large favorable entropy increase but with an unfavorable enthalpy increase in the pH range of 3.5-5.5 and at all temperatures examined. Aluminum ions show a strong and pH-dependent binding affinity to CT DNA, and a large positive molar heat capacity change for the binding, 1.57 kcal mol 1K 1, demonstrates the burial of the polar surface of CT DNA upon groove binding. The fluorescence of ethidium bromide bound to CT DNA is quenched by aluminum ions in a dynamic way. Both Stern-Volmer quenching constant and the binding constant increase with the increase of the pH values, reaching a maximum at pH 4.5, and decline with further increasing the pH to 5.5. At pH 6.0 and 7.0, aluminum ions precipitate CT DNA completely and no binding of aluminum ions to CT DNA is observed by ITC. Combining the results from these three methods, we conclude that aluminum ions bind to CT DNA with high affinity through groove binding under aluminum toxicity pH conditions and precipitate CT DNA under physiological conditions.