Kakabakos S E, Tyllianakis P E, Evangelatos G P, Ithakissios D S
Institute of Radioisotopes and Radiodiagnostic Products, Radioimmunochemistry Laboratory, NCSR Demokritos, Athens, Greece.
Biomaterials. 1994 Mar;15(4):289-97. doi: 10.1016/0142-9612(94)90054-x.
A simple and sensitive method for the quantitative determination of solid-supported primary and/or secondary amino groups using commercially available reagents is described. The solid supports are treated in an aqueous environment with either 2-iminothiolane (ITL) or sulpho-succinimidyl-3-(4-hydroxyphenyl)propionate (sulpho-SHPP), which introduce one sulphydryl or one hydroxyphenyl group per amino group reacted, respectively. These groups are capable of reducing Cu2+ to Cu+ in alkaline medium. Thus, after removal of the excess reagents through washing, subsequent incubation of the solids with 2,2'-bicinchoninic acid (BCA) copper protein reagent results in production of Cu+ in the solution, which forms a chelate complex with BCA absorbing at 562 nm. The quantitation of the groups introduced on the surfaces, and therefore of the reacted amino groups, is carried out through standard curves of cysteine solutions for ITL, or tyrosine solutions for sulpho-SHPP-treated solids. Using ITL, only the primary amino groups are determined, whereas sulpho-SHPP provided the primary and secondary reactive amino groups. The method is versatile and can be used for the estimation of amino groups onto several biomedical solid matrices, and should provide useful information for the covalent immobilization of ligands (e.g. drugs, antibodies).
本文描述了一种使用市售试剂定量测定固相支持物上伯氨基和/或仲氨基的简单灵敏方法。固相支持物在水性环境中用2-亚氨基硫杂环戊烷（ITL）或磺基琥珀酰亚胺-3-(4-羟基苯基)丙酸酯（磺基-SHPP）处理，它们分别与每个反应的氨基引入一个巯基或一个羟基苯基。这些基团能够在碱性介质中将Cu2+还原为Cu+。因此，通过洗涤除去过量试剂后，随后将固相与2,2'-二喹啉甲酸（BCA）铜蛋白试剂孵育，会在溶液中产生Cu+，其与BCA形成螯合物并在562 nm处有吸收。通过ITL的半胱氨酸溶液标准曲线或磺基-SHPP处理的固相的酪氨酸溶液标准曲线，对引入表面的基团进行定量，从而对反应的氨基进行定量。使用ITL时，只能测定伯氨基，而磺基-SHPP可测定伯氨基和仲反应性氨基。该方法用途广泛，可用于评估多种生物医学固体基质上的氨基，应为配体（如药物、抗体）的共价固定提供有用信息。
