Direct Colorimetric Determination of Solid-Supported Functional Groups and Ligands Using Bicinchoninic Acid

Anal Biochem. 1994 Jun;219(2):335-40. doi: 10.1006/abio.1994.1273.

Abstract

We describe new colorimetric methods for the direct determination of total solid-supported sulfydryl, aldehydo, hydrazido, and N-hydroxysuccinimido carboxylate groups as well as of immobilized cysteine, tyrosine, and thyroxin using only the commercially available bicinchoninic acid/copper protein assay reagent. The method is based on the ability of these groups to reduce Cu2+ to Cu+, which forms a chelate complex with bicinchoninic acid absorbing at 562 nm. Each assay requires only one 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 through standard curves of appropriate substances. Using the assays developed we determined the amount of the above-mentioned functional groups and ligands onto several commercially available solid supports. The values obtained were in agreement with those provided by relative literature methods and/or by the manufacturers. The assays were found to be accurate, precise (interassay CV less than 3%), and very sensitive, allowing the determination of nmol quantities of functional groups per assay tube.

Publication types

  • Comparative Study

MeSH terms

  • Aldehydes / analysis*
  • Amino Acids / analysis*
  • Carboxylic Acids / analysis*
  • Colorimetry / methods*
  • Cysteine / analysis
  • Hydrazines / analysis*
  • Indicators and Reagents
  • Ligands
  • Quinolines*
  • Sulfhydryl Compounds / analysis*
  • Thyroxine / analysis*
  • Tyrosine / analysis

Substances

  • Aldehydes
  • Amino Acids
  • Carboxylic Acids
  • Hydrazines
  • Indicators and Reagents
  • Ligands
  • Quinolines
  • Sulfhydryl Compounds
  • Tyrosine
  • bicinchoninic acid
  • Cysteine
  • Thyroxine