Garel J R
Eur J Biochem. 1976 Nov 1;70(1):179-89. doi: 10.1111/j.1432-1033.1976.tb10968.x.
A chemical derivative of bovine pancreatic ribonuclease A (RNase A) has been prepared by reaction with fluorescein-isothiocyanate at pH 6. This derivative has a fluorescein group covalently attached to the alpha-amino group of the protein. The enzymic properties of the modified protein are similar to those of RNase A. It is shown that the pK of the fluorescein group can be used as an index of protein conformation to monitor structural changes in the protein. In this work, the binding of a specific inhibitor (cytidine 2'-monophosphate) to RNase A, the isomerization process occurring in RNase A around pH 6, and the thermal unfolding of RNase A, were studied by mean of the pK changes of the fluorescein group. The results obtained by this method are fully consistent with those obtained by other methods. It is proposed that using ionizable reporter groups and their changes in pK to monitor conformational changes in proteins may be a sensitive tool both in equilibrium and kinetic studies.
通过在pH 6条件下与异硫氰酸荧光素反应制备了牛胰核糖核酸酶A（RNase A）的一种化学衍生物。该衍生物有一个荧光素基团共价连接到蛋白质的α-氨基上。修饰后蛋白质的酶学性质与RNase A相似。结果表明，荧光素基团的pK可作为蛋白质构象指标来监测蛋白质的结构变化。在本研究中，通过荧光素基团pK的变化研究了特异性抑制剂（2'-单磷酸胞苷）与RNase A的结合、RNase A在pH 6左右发生的异构化过程以及RNase A的热解折叠。用该方法得到的结果与用其他方法得到的结果完全一致。有人提出，利用可电离的报告基团及其pK变化来监测蛋白质的构象变化，在平衡和动力学研究中可能是一种灵敏的工具。
