DISULFIDE SCRAMBLING OF INTERLEUKIN-2 - HPLC RESOLUTION OF THE 3 POSSIBLE ISOMERS
DISULFIDE SCRAMBLING OF INTERLEUKIN-2 - HPLC RESOLUTION OF THE 3 POSSIBLE ISOMERS
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DOI：
10.1016/0003-2697(86)90236-8
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发表时间：
1986-05-15
影响因子：
2.9
通讯作者：
SMART, JE
中科院分区：
文献类型：
--
作者：
BROWNING, JL;MATTALIANO, RJ;SMART, JE
天然白细胞介素-2 （IL-2）含有三种半胱氨酸；两个存在于二硫桥中（Cys-58和Cys-105），第三个Cys-125是一个游离的巯基。在碱性条件下，6 M胍盐酸盐存在时，IL-2转化为3个异构体。每个异构体代表三种可能的二硫化物连接形式之一，可以由三种半胱氨酸产生。这三种异构体在C4反相高效液相色谱系统上进行了分离。通过用[3H]碘乙酸对各同分异构体中的游离半胱氨酸进行羧甲基化，然后通过色氨酸图测定标记的半胱氨酸，确定了三种形式中的每一种形式的身份。两个混乱峰中较占优势的色氨酸图谱显示它是IL-2的Cys-105-S-S-Cys-125连锁形式。Ser-125构建的IL-2，缺乏游离半胱氨酸，在这些条件下没有抢夺。这些实验证明了反相高效液相色谱在蛋白质折叠和二硫键结构研究中的实用性。
Native interleukin-2 (IL-2) contains three cysteines; two exist in a disulfide bridge (Cys-58 and Cys-105) and the third Cys-125 is a free sulfhydryl. In the presence of 6 M guanidine hydrochloride at alkaline pH, IL-2 is converted into three isomers. Each isomer represents one of the three possible disulfide-linked forms that can be generated from three cysteines. These three isomers were resolved on a C4 reverse-phase HPLC system. The identity of each of the three forms was determined by carboxymethylation of the free cysteines in each isomer with [3H]iodoacetic acid followed by determination of the labelled cysteines by tryptic peptide mapping. Tryptic peptide mapping of the more predominant of the two scrambled peaks showed it to be the Cys-105-S-S-Cys-125 linked form of IL-2. A Ser-125 construction of IL-2, which lacks a free cysteine, did not scramble under these conditions. These experiments demonstrate the utility of reverse-phase HPLC in studies of protein folding and disulfide bond structure.