Study of pH and temperature-induced transitions in urate oxidase (Uox-EC1.7.3.3) by microcalorimetry (DSC), size exclusion chromatography (SEC) and enzymatic activity experiments.

Purified recombinant urate oxidase (urate oxygen oxidoreductase EC 1.7.3.3. re-Uox) has been studied by means of differential scanning calorimetry (DSC) in correlation with enzymatic activity measurements and size exclusion chromatography. Differential scanning calorimetry curves versus pH show two endothermal effects in the pH range 6-10. The first endotherm reveals a maximum stability between pH 7.25 and pH 9.5 corresponding to a temperature of transition T(m1) of 49.0 degrees C and an enthalpy of transition of 326 kJ mol(-1). This value dramatically decreases below pH 7.25. The behavior of the second endotherm is more complex but the temperature of transition T(m2) is constant between pH 9 and 7.25 and a maximum for the corresponding enthalpy is obtained near pH 8 with DeltaH(2)=272 kJ mol(-1). An optimal pH of 8.0 for the stability of the enzymatic activity at elevated temperature was also found which was in good agreement with calorimetric results. Reversibility of the first endotherm is obtained from 20 to 51.5 degrees C. The calorimetric result is correlated to enzymatic activity, purity by size exclusion chromatography (SEC) and protein concentration measurements. In contrast, for the second endotherm, after heating up to 68.9 degrees C, no reversibility was found. Interaction with structural analogues of urate has been studied by DSC. 8-Azahyooxanthine has only a small effect and caffeine has no effect at all. With 8-azaxanthine, a rapid increase of the T(m1) function of the concentration is obtained. At high concentration T(m1) reached the T(m2) value which remained unaffected.

Involvement of protein solvation in the interaction between a contrast medium (iopamidol) and fibrinogen or lysozyme.

The interaction between proteins and a radiological commonly-used contrast medium (iopamidol) have been studied by calorimetry. When aqueous solutions of fibrinogen or of lysozyme (20 g/l) are mixed with an aqueous solution of iopamidol (1,3-5 benzendicardoxamid,N,N'-bis[2-hydroxy-1-(hydroxymethyl)ethyl]-5- [(2-hydroxy-1-oxopropyl)amino]-2,4,6-triiodo) in the clinical blood concentration range (26-485 mM), isothermal calorimetry reveals a weak endothermal interaction at a high concentration of iopamidol for both proteins. This endothermal effect does not appear to be due to direct protein-iopamidol association. Differential scanning calorimetry confirms the influence of iopamidol by the change in protein unfolding in the presence of contrast medium, and suggests alterations in the protein solvation as a mechanism. Dilution studies indicate that iopamidol can influence protein solvation even when water molecules are present in a molecular excess of 1000. The influence of iopamidol on the availability of water molecules and the absence of direct interaction with the protein molecules is shown by Raman spectroscopy of two amino acids in the presence of iopamidol. The spectrum of alanine is unchanged at any iopamidol concentration studied, whereas the spectrum lines due to the thiol group of cysteine are shifted in a manner consistent with altered solvation.

Phase separation of miscible phospholipids by sonication of bilayer vesicles.

Sonication of phospholipid vesicles may result, according to their liquid or solid crystal state, in the generation of unilamellar vesicles or structural defects within their bilayers, respectively. The transition temperature Tm of the phospholipid bilayer is usually the threshold temperature delineating the physical effects of ultrasound. However, for vesicles made from a mixture of two miscible phospholipids, this threshold temperature was not found to be the intermediate Tm of the phospholipid mixture bilayers, but the Tm of the lowest melting component. This was due to a simultaneous lateral phase separation of the two phospholipids induced by the sonication as demonstrated by differential scanning calorimetry analysis.