CONJUGATION TO PREACTIVATED PROTEINS USING DIVINYLSULFONE AND IODOACETIC ACID
CONJUGATION TO PREACTIVATED PROTEINS USING DIVINYLSULFONE AND IODOACETIC ACID
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DOI：
10.1016/0022-1759(94)00345-w
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发表时间：
1995-04-26
影响因子：
2.2
通讯作者：
JENSEN, OM
中科院分区：
文献类型：
--
作者：
HOUEN, G;JENSEN, OM
提出了两种温和条件下蛋白质预活化和多肽与蛋白质偶联的方法。蛋白质与二乙烯基砜(DVS)的预活化允许多肽通过氨基、羟基或巯基结合，这取决于所使用的偶合pH，而用碘乙酸(IAA)N-羟基琥珀酰亚胺酯的预活化允许通过巯基选择性结合。此外，后一种方法可以通过测定酸解后的羧甲基半胱氨酸来定量偶联率。二乙烯基砜活化蛋白可以在-20℃下长时间保存，直到需要结合，而碘乙酸活化蛋白可以在-20℃下保存几天。为了获得用于免疫目的和用作免疫分析试剂的肽/蛋白质结合物，研究了这些结合方法。DVS激活的蛋白质允许黄体释放激素(LHRH)通过其酪氨酸侧链直接结合，并允许合成明确的连接物。比较了牛血清白蛋白(BSA)、还原和羧甲基化牛血清白蛋白(BSA)和纯化蛋白衍生物(PPD)作为载体获得LHRH(M(R)1000)和EGF(M(R)5000)抗体的潜在价值。IAA-PPD被评价为通过半胱氨酸侧链特异性结合谷胱甘肽的载体和获得谷胱甘肽抗体的载体。获得的抗血清具有特异性和高滴度，因此这里描述的方法将允许方便地合成具有明确特征的载体结合物。
Two methods for the preactivation of proteins and conjugation of peptides to proteins under mild conditions are presented. Preactivation of proteins with divinylsulfone (DVS) permits peptide conjugation through either amino, hydroxyl or sulphydryl groups depending on the coupling pH used, while preactivation with iodoacetic acid (IAA) N-hydroxy-succinimide ester permits selective conjugation through sulphydryl groups. In addition, the latter method allows quantitation of the conjugation ratio through determination of carboxymethyl cysteine after acid hydrolysis. The divinylsulfone activated proteins can be stored for extended periods of time at -20 degrees C until required for conjugation, while the iodoacetic acid activated protein can be stored for a few days at -20 degrees C. These conjugation methods were investigated with respect to obtaining peptide/protein conjugates for immunization purposes and for use as reagents in immunoassays. The DVS activated proteins permitted direct conjugation of luteinizing releasing hormone (LHRH) through its tyrosine side chain and allowed synthesis of well defined conjugates. The DVS derivatives of bovine serum albumin (BSA), reduced and carboxymethylated BSA and purified protein derivative (PPD) were compared with respect to their potential value as carriers for obtaining antibodies to LHRH (M(r) 1000) and epidermal growth factor (EGF, M(r) 5000). IAA-PPD was evaluated as a carrier for the conjugation of glutathione specifically through its cysteine side chain and for obtaining antibodies to glutathione. The antisera obtained were specific and of high titer, and the methods described here will thus allow the convenient synthesis of carrier conjugates with well defined characteristics.