Highly selective assay of proteins in dilute solutions

Abstract

A very simple, highly selective assay of proteins has been developed. Solutions of 6 M hydrochloric acid containing 0–20 μg ml −1 protein are subjected to acid hydrolysis, in the presence of air, during their evaporation to dryness on a steam bath. Then, the resulted amino acids are determined with a 2,2-dihydroxy-1,3-indanedione reagent. To eliminate any interference and to increase in selectivity of the assay, a blank with the unhydrolyzed protein is carried out. The protein being analyzed or alanine is used to plot calibration curves. The absorbance of the colored solution is read at 516 nm. The color system obeys Beer's law in the range 0.1–20 μg ml −1 protein. The molar absorptivity for alanine was found to be 1.69×10 3 (±9.7) l mol −1 cm −1. Also, the relative molar absorptivity for peptone was found to be 1.23×103 (±6.5) l mol −1 cm −1. The lowest limits for the estimation of serum albumin, gelatin, and peptone are around 0.25, 0.19 and 0.31 μg ml −1, respectively. The method is highly selective and the determination is little affected by the presence of other substances. All other important analytical parameters were studied and practical applicability of the method has been verified by the analysis of some biological materials.