Direct colorimetric determination of solid-supported functional groups and ligands using bicinchoninic acid.
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.
Tyllianakis
P E
PE
Institute of Radioisotopes and Radiodiagnostic Products, N.C.S.R. Demokritos, Athens, Greece.
Kakabakos
S E
SE
Evangelatos
G P
GP
Ithakissios
D S
DS
D000447:Aldehydes / Q000032:analysis*; D000596:Amino Acids / Q000032:analysis*; D002264:Carboxylic Acids / Q000032:analysis*; D003124:Colorimetry / Q000379:methods*; D003545:Cysteine / Q000032:analysis; D006834:Hydrazines / Q000032:analysis*; D007202:Indicators and Reagents; D008024:Ligands; D011804:Quinolines; D013438:Sulfhydryl Compounds / Q000032:analysis*; D013974:Thyroxine / Q000032:analysis*; D014443:Tyrosine / Q000032:analysis
D000447:Aldehydes; D000596:Amino Acids; D002264:Carboxylic Acids; D006834:Hydrazines; D007202:Indicators and Reagents; D008024:Ligands; D011804:Quinolines; D013438:Sulfhydryl Compounds; D014443:Tyrosine; C047117:bicinchoninic acid; D003545:Cysteine; D013974:Thyroxine