Fatima Shamila, Jairajpuri Deeba S, Saleemuddin M
Department of Biochemistry, Aligarh Muslim University, Aligarh 202002, India.
J Biochem Biophys Methods. 2008 Apr 24;70(6):958-65. doi: 10.1016/j.jprot.2007.10.002. Epub 2007 Nov 12.
A procedure for the rapid screening of inhibitors of glycation reaction, based on their ability to protect RNase against sugar induced inactivation of the enzyme is described. Glycation is implicated in variety of disorders including diabetes, atherosclerosis various micropathies yet is a slow process both in vivo and in vitro. In order to speed up glycation, the reaction was carried out at 60 degrees C using a thermostable protein RNase and ribose, a sugar that is known to react rapidly than glucose in the glycation reaction. It was observed that incubation of RNase with ribose at 60 degrees C in rapid inactivation of the enzyme with a parallel decrease in tyrosine fluorescence, enhancement in new fluorescence and hyperchromicity in the UV-region. No such alterations in the enzyme activity were observed when the incubation was carried out in absence of the sugar. Compounds and drugs that are known to act as inhibitors of glycation reaction restricted the ribose-induced inactivation of RNase. RNase immobilized on CNBr-activated Sepharose was also sensitive to exposure to ribose and appeared a better system to screen inhibitors of glycation from natural sources that contain substances that interfere with the assay of enzyme as well as in the study of post Amadori inhibitors of glycation.
本文描述了一种基于抑制剂保护核糖核酸酶（RNase）免受糖诱导的酶失活能力的快速筛选糖基化反应抑制剂的方法。糖基化与多种疾病有关，包括糖尿病、动脉粥样硬化和各种微血管病变，但在体内和体外都是一个缓慢的过程。为了加速糖基化反应，使用热稳定蛋白核糖核酸酶和核糖在60℃下进行反应，核糖是一种已知在糖基化反应中比葡萄糖反应更快的糖。观察到核糖核酸酶在60℃下与核糖孵育会导致酶快速失活，同时酪氨酸荧光平行下降，新荧光增强，紫外区域出现增色现象。在无糖的情况下进行孵育时，未观察到酶活性有此类变化。已知作为糖基化反应抑制剂的化合物和药物可限制核糖诱导的核糖核酸酶失活。固定在溴化氰活化琼脂糖上的核糖核酸酶对核糖暴露也敏感，似乎是一种更好的系统，可用于从含有干扰酶测定物质的天然来源中筛选糖基化抑制剂，以及用于研究阿马多里反应后糖基化抑制剂。
