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 calori metry (DSC) in correlation with enzymatic activity measurements and si ze 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 corr esponding 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 dec reases below pH 7.25. The behavior of the second endotherm is more com plex but the temperature of transition T-m2 is constant between pH 9 a nd 7.25 and a maximum for the corresponding enthalpy is obtained near pH 8 with Delta H-2 = 272 kJ mol(-1). An optimal pH of 8.0 far the sta bility of the enzymatic activity at elevated temperature was also foun d which was in good agreement with calorimetric results. Reversibility of the first endotherm is obtained from 20 to 51.5 degrees C. The cal orimetric result is correlated to enzymatic activity, purity by size e xclusion chromatography (SEC) and protein concentration measurements. In contrast, for the second endotherm, after heating up to 68.9 degree s C, no reversibility was found. Interaction with structural analogues of urate has been studied by DSC. 8-Azahypoxanthine 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 hig h concentration T-m1 reached the T-m2 value which remained unaffected.