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首页> 外文期刊>Biophysical Chemistry: An International Journal Devoted to the Physical Chemistry of Biological Phenomena >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
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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

机译：微量热法（DSC），排阻色谱法（SEC）和酶活性实验研究尿酸氧化酶（UOX-EC1.7.3.3）中pH和温度诱导的转变

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摘要

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 Delta H-2 = 272 kJ mol(-1). An optimal pH of 8.0 far 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-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 high concentration T-m1 reached the T-m2 value which remained unaffected. [References: 13]
机译：纯化的重组尿酸氧化酶（尿酸氧氧化还原酶EC 1.7.3.3 re-Uox）已通过差示扫描量热法（DSC）与酶活性测量和尺寸排阻色谱法进行了研究。差示扫描量热曲线与pH的关系曲线表明在pH 6-10范围内有两种吸热作用。第一次吸热揭示了在pH 7.25和pH 9.5之间的最大稳定性，对应于49.0摄氏度的转变温度T-m1和326 kJ mol（-1）的转变焓。该值在pH 7.25以下急剧降低。第二个吸热的行为更为复杂，但转变温度T-m2在pH 9和7.25之间保持恒定，并且在pH 8附近获得了相应的焓的最大值，ΔH-2 = 272 kJ mol（-1）。还发现最佳pH 8.0远高于高温下酶促活性的稳定性，这与量热法结果非常吻合。第一次吸热的可逆性是在20至51.5摄氏度下获得的。量热结果与酶活性，尺寸排阻色谱法（SEC）的纯度和蛋白质浓度的测量值相关。相反，对于第二个吸热，在加热至68.9℃之后，没有发现可逆性。 DSC已经研究了与尿酸盐的结构类似物的相互作用。 8-氮杂黄嘌呤仅具有很小的作用，咖啡因根本没有作用。使用8-azaxanthine，可以快速提高浓度的T-m1函数。在高浓度下，T-m1达到不受影响的T-m2值。 [参考：13]

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