COLORIMETRIC DETERMINATION OF REACTIVE SOLID-SUPPORTED PRIMARY AND SECONDARY AMINO-GROUPS

A simple and sensitive method for the quantitative determination of so lid-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-succi nimidyl-3-(4-hydroxyphenyl)propionate (sulpho-SHPP), which introduce o ne sulphydryl or one hydroxyphenyl group per amino group reacted, resp ectively. These groups are capable of reducing Cu2+ to Cu+ in alkaline medium. Thus, after removal of the excess reagents through washing, s ubsequent incubation of the solids with 2,2'-bicinchoninic acid (BCA) copper protein reagent results in production of Cu+ in the solution, w hich forms a chelate complex with BCA absorbing at 562 nm. The quantit ation of the groups introduced on the surfaces, and therefore of the r eacted amino groups, is carried out through standard curves of cystein e solutions for ITL, or tyrosine solutions for sulpho-SHPP-treated sol ids Using ITL, only the primary amino groups are determined, whereas s ulpho-SHPP provided the primary and secondary reactive amino groups. T he method is versatile and can be used for the estimation of amino gro ups onto several biomedical solid matrices, and should provide useful information for the covalent immobilization of ligands (e.g. drugs, an tibodies).