Spectrophotometric total protein assay with copper(II)-neocuproine reagent in alkaline medium

Abstract

Total protein assay was made using copper(II)-neocuproine (Nc) reagent in alkaline medium (with the help of a hydroxide-carbonate-tartarate solution) after 30min incubation at 40 degrees C. The absorbance of the reduction product, Cu(I)-Nc complex, was recorded at 450nm against a reagent blank. The absorptivity of the developed method for bovine serum albumin (BSA) was 0.023lmg(-1)cm(-1), greater than that of Lowry assay (0.0098), and much greater than that of Cu(II)-bicinchoninic acid (BCA) assay (0.00077). The linear range of the developed method (8-100mgl(-1) BSA) was as wide as that of Lowry, and much wider than that of BCA (200-1000mgl(-1) BSA) assay. The sensitivity of the method was greater than those of Cu-based assays (biuret, Lowry, and BCA) with a LOD of 1mgl(-1) BSA. The within-run and between-run precisions as RSD were 0.73 and 1.01%, respectively. The selectivity of the proposed method for protein was much higher than those of dye-binding and Lowry assays: Most common interferents to other protein assays such as tris, ethanolamine, deoxycholate, CsCl, citrate, and triton X-100 were tolerated at 100-fold concentrations in the analysis of 10mgl(-1) BSA, while the tolerance limits for other interferents, e.g., (NH(4))(2)SO(4) and acetylsalicylic acid (50-fold), SDS (25-fold), and glycerol (20-fold) were at acceptable levels. The redox reaction of Cu(II)-Nc as an outer-sphere electron transfer agent with the peptide bond and with four amino acid residues (cystine, cysteine, tryptophan, and tyrosine) was kinetically more favourable than that of Cu(II) alone in the biuret assay. Since the reduction product of Cu(II) with protein, i.e., Cu(I), was coordinatively saturated with Nc in the stable Cu(Nc)(2)(+) chelate, re-oxidation of the formed Cu(I) with Fenton-like reactions was not possible, thereby preventing a loss of chromophore. After conventional protein extraction, precipitation, and redissolution procedures, the protein contents of the minced meat (veal and turkey), sardine, various milk products, and egg white were analyzed with the proposed and Lowry methods, and the results correlated appreciably (r=0.98). The method was validated by Kjeldahl analyses of the tested samples; the data sets of complex samples assayed by Cu(II)-Nc and Lowry correlated to the findings of Kjeldahl yielded correlation coefficients r=0.96 and 0.97, respectively, with slopes being close to 1. Interferences of glucose and thiol compounds at relatively low concentrations could be compensated for by selecting a lower alkaline pH (i.e., pH 10) at a cost of slightly reduced sensitivity and adding an identical amount of interferent to the reagent blank, respectively, since the absorbances due to BSA and interferent were additive. Thus a novel spectrophotometric method for total protein assay using a stable reagent and chromophore, which was simple, rapid, sensitive, flexible, and relatively selective, was developed, and applied to a variety of food products.