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

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).