MEASUREMENT OF THE REACTIVE AND AVAILABLE SOLID-SUPPORTED CARBOXYLIC,N-HYDROXYSUCCINYLATED CARBOXYLIC, AND ALDEHYDO GROUPS

We describe new colorimetric methods for the determination of the reac tive and available solid-supported carboxylic, N-hydroxysuccinylated c arboxylic, and aldehydo groups under conditions usually applied for th e coupling of biomolecules. The methods involve the reaction of the so lid-supported functional groups with tyramine or cysteine, and the sub sequent titration of the ligand coupled onto the solid supports using the commercially available bicinchoninic acid/copper protein assay rea gent (BCA). The titration is based on the ability of these ligands to reduce Cu2+ to Cu+, which forms a chelate complex with bicinchoninic a cid absorbing at 562 nm. The quantitation is finally carried out throu gh standard curves obtained using tyramine or cysteine solutions of kn own concentrations. The values obtained by the assays developed for se veral solid supports carrying carboxylic, NHS-ester, and aldehydo grou ps were well correlated with those obtained by other literature method s or provided by the manufacturers. All of the proposed methods are si mple, more sensitive than other relevant literature methods, and requi re only commericially available reagents.