DIRECT COLORIMETRIC DETERMINATION OF SOLID-SUPPORTED FUNCTIONAL-GROUPS AND LIGANDS USING BICINCHONINIC ACID

We describe new colorimetric methods for the direct determination of t otal solid-supported sulfhydryl, aldehydo, hydrazido, and N-hydroxysuc cinimido carboxylate groups as well as of immobilized cysteine, tyrosi ne, and thyroxin using only the commercially available bicinchoninic a cid/copper protein assay reagent. The method is based on the ability o f these groups to reduce CU2+ to Cu+, which forms a chelate complex wi th bicinchoninic acid absorbing at 562 nm. Each assay requires only on e incubation step of the solids with the reagent for 1 h at 60 degrees C. The quantitation of the different groups is finally carried out th rough standard curves of appropriate substances. Using the assays deve loped we determined the amount of the above-mentioned functional group s and ligands onto several commercially available sold supports. The v alues obtained were in agreement with those provided by relative liter ature methods and/or by the manufacturers. The assays were found to be accurate, precise (interassay CV less than 3%), and very sensitive, a llowing the determination of nmol quantities of functional groups per assay tube. (C) 1994 Academic Press,Inc.