Jose M. Sanchez-Ruiz

Differential Scanning Calorimetry of Proteins

Differential scanning calorimetry (DSC) is a powerful technique to characterize temperature-induced conformational changes in proteins and other biological macromolecules. In fact, DSC studies on protein thermal denaturation have played a central role in the development of current views about the factors that determine protein stability. Reviews on the various aspects of this technique are available in the literature (see, for instance, Privalov, 1979, 1982, 1989; Mateo, 1984; Sturtevant, 1987; Sanchez-Ruiz and Mateo, 1987; Freire et al., 1990) and, consequently, this chapter will mainly focus on recent developments in the field. The relevant features of the DSC experiment and the preliminary data analysis will be outlined in this section, and the well-known equilibrium thermodynamics procedures for the analysis of the DSC profiles will be briefly summarized in Section 2.

Rates and equilibria of aldimine formation between pyridoxal 5′-phosphate and N-hexylamine

The rate and equilibrium constants of the reaction between n-hexylamine and pyridoxal 5′-phosphate have been studied in aqueous solution as a function of pH at 25 ± 0.1 °C and at an ionic strength of 0.1. The pH profiles of the observed constants have been explained by protonation of the pyridoxal 5′-phosphate and its Schiffs base. This study shows that there is an intramolecular general acid catalysis of the Schiffs base formation, which can explain why the aldimine is formed at a reasonably high rate at neutral pH, in spite of the low amount of unprotonated reacting amine at this pH.

Thermodynamic constants for tautomerism and ionization of pyridoxine and 3-hydroxypyridine in water–dioxane

Thermodynamic values of the ionization constants and tautomeric equilibrium constants of pyridoxine and 3-hydroxypyridine were determined in dioxane–water at 25 °C. The Born model is unable to explain the effect of the solvent on these constants. The logarithms of the equilibrium constants calculated change linearly according to the logarithm of the molar water content in the mixture, up to 40% dioxane in the case of pyridine ionizations, and up to 70% dioxane (maximum content investigated) for the phenol ionizations. The effect of protonation of either the phenolate or pyridinium nitrogen groups on the pK of the other group in these substances increases when the water content in the medium is decreased.

A calorimetric approach to the kinetics of the irreversible thermal denaturation of proteins

Abstract Differential scanning calorimetry has been used to characterize the thermal denaturation of three proteins: porcine pancreatic carboxypeptidase B, bovine pancreatic α-chymotrypsin and bacteriorhodopsin from the purple membrane of Halobacterium halobium . The corresponding thermal transitions were found to be irreversible and rate-limited under some experimental conditions. Kinetic models based on the scan-rate effect on the thermograms have been applied to analyse these irreversible denaturation processes.

Thermodynamic equilibrium constants for pyridoxal and pyridoxal 5′-phosphate in dioxane–water mixtures

The electronic absorption spectra of pyridoxal and pyridoxal 5′-phosphate have been obtained in water–dioxane mixtures (0–70% v/v) at 25 °C, and resolved using log–normal curves. The thermodynamic values of the hydration, hemiacetal, and tautomeric equilibrium constants together with the microscopic pK values for these compounds in all the mixtures studied could then be obtained. The variation of these pK values according to solvent polarity fits Marshalls model satisfactorily.

The equilibria of 5-deoxypyridoxal in water–dioxane mixtures

The electronic absorption spectra of 5-deoxypyridoxal have been obtained together with its macroscopic pK values in water-dioxane solution at 25 °C. These spectra have been resolved using log-normal curves, enabling the determination of the spectra of the formyl, hydrated, and tautomeric forms. The proportions of these forms could thus be ascertained and numerical values for the hydration and tautomeric equilibrium constants obtained. The microscopic protonation constants have been calculated from these equilibrium constants and from the macroscopic pK values, which were determined potentiometrically. The variation of these equilibrium constants according to solvent polarity fits Marshalls model satisfactorily.

Enthalpy, Entropy and Gibbs Energy Changes Associated with the Ionization of Vitamin B6 Molecules in Water-Dioxane Systems

The thermodynamic macroscopic ionization, tautomeric and hydration constants of several vitamin B6, molecules have been measured as a function of the temperature and solvent composition in water-dioxane mixtures. From these values the microscopic equilibrium constants for the ionization of the 3-hydroxy (pKOH) and nitrogen pyridinium (pKNH) groups of these molecules have been obtained, as well as their ΔH and ΔS° values. The AH values are practically independent of the solvent composition, while the AS values clearly depend on this composition. The solvent influence on the ΔG, or pK values, is thus of an entropic origin, probably arising from changes in the order of the water solvation molecules.