Tyllianakis P E, Kakabakos S E, Evangelatos G P, Ithakissios D S
Institute of Radioisotopes and Radiodiagnostic Products, N.C.S.R. Demokritos, Athens, Greece.
Anal Biochem. 1994 Jun;219(2):335-40. doi: 10.1006/abio.1994.1273.
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.
我们描述了仅使用市售的二辛可宁酸/铜蛋白测定试剂直接测定总固体支持的巯基、醛基、酰肼基和N-羟基琥珀酰亚胺羧酸酯基团以及固定化的半胱氨酸、酪氨酸和甲状腺素的新比色法。该方法基于这些基团将Cu2+还原为Cu+的能力，Cu+与在562 nm处有吸收的二辛可宁酸形成螯合物。每次测定仅需将固体与试剂在60℃下孵育1小时。最后通过适当物质的标准曲线对不同基团进行定量。使用所开发的测定方法，我们测定了几种市售固体支持物上上述官能团和配体的量。获得的值与相关文献方法和/或制造商提供的值一致。发现这些测定准确、精确（批间CV小于3%）且非常灵敏，每次测定管可测定纳摩尔量的官能团。
