Interference of Anthracycline Derivatives with Measurement of Proteins with BCA

Common methods for the assay of proteins in biological fluids include Lowry (1), Coomassie Brilliant Blue G-250 protein dye-binding assay (Bradford) (2), and the 2,2′-bicinchoninic acid (BCA) assay (3). Among them, the Lowry method has been used widely for protein quantification in biological samples. However, many substances commonly used during protein purification interfere with the Lowry assay. To overcome this, the BCA method was developed. This assay is similar to the Lowry method in that both rely on the biuret reaction for generation of a colored complex between peptide bonds and cuprous ions when protein is placed in an alkaline environment containing Cu2+. Unlike the Lowry method, the BCA method has been found to have exceptional tolerance to nonionic detergents and simple buffer salts. However, sucrose and detergents, biogenic amines, chlorpromazine, penicillins, vitamin C, paracetamol, hydrogen peroxides, or any compound that can reduce Cu2+ to Cu+ will produce the characteristic purple color associated with the binding of Cu+ with BCA and interfere with this method (4)(5). It is not surprising that anthracyclines, which are easily oxidized in alkaline media in the presence of metal ions, would produce the necessary reduction of Cu2+ for the formation of a Cu+-BCA complex. In the Bradford method, the Coomassie blue dye binds to primarily basic and aromatic amino acid residues, especially arginine, and forms colored complexes. In this method, interferences may be caused by drug–protein and (or) drug–dye interactions. Because the mechanism of color formation is different in the BCA and the Bradford methods, both methods were considered for interference studies. The primary objective of this report was to determine whether an anthracycline, e.g., doxorubicin (Dox), may interfere with the measurement of protein concentrations with the BCA and the Bradford assays. We examined the sensitivity and specificity of the BCA and Coomassie Brilliant Blue for Dox in this study. Such findings may have significant implications in the assay of protein when an anthracycline is present. Dox showed a significant interference in the BCA assay, whether assayed in the presence or absence of a bovine serum albumin (BSA) protein calibrator (Fig. 1 ). The interference was found to be linear over the standard protein range, with an immediate appearance of typical assay color without turbidity (even after further dilutions). The reaction of Dox with the BCA reagent at room temperature (RT) for 2 h suggested that a wide range of anthracyclines will react with the BCA reagent to produce the characteristic purple color if the reduction of Cu2+ to Cu+ by anthracyclines is dependent on the anthracycline nucleus rather than on the side-chain substitution. The detection limits were 2 × 10−4 g/L for Dox and 6 × 10−3 g/L for BSA. Although protein shows a nonlinear color response with BCA with increasing concentration, at low concentration the response is linear (Fig. 1 ). Dox did not interfere with the Bradford method to quantify protein (not shown). The development of color associated with the reaction of protein with the BCA reagent is slow at RT and enhanced by increasing the temperature and incubation time (3). The color developed after 2 h at RT is similar to that developed over 30 min at 37 °C. Dox concentrations of 0.01 to 0.10 g/L with different protein concentrations have produced color intensities that are significantly higher than those due to the protein alone (data not shown). These results indicate that anthracyclines will significantly enhance color development produced by proteins at anthracycline concentrations that exceed 2 × 10−4 g/L per gram of protein. Measurement of the total protein concentration of the plasma used to monitor the nutritional status of patients receiving chemotherapy (6), and changes in serum and urine protein may reflect chemotherapy-associated nephrotoxicity (7). When calculated for a healthy individual, the plasma concentration of Dox or its metabolites is expected to be >35 mg/L, as >75% of Dox is bound to plasma proteins (8)(9). Therefore, plasma concentrations of anthracyclines, including both the parent and the metabolites, are expected to be >0.2 μg/mg of protein. At this low drug-to-protein ratio Dox would not increase the protein concentration significantly when measured by the BCA method. Anthracycline derivatives have been widely used in experimental therapeutics. For example, anthracyclines such as daunomycin have been loaded into lipoproteins and their distribution in plasma proteins has been studied (10). To quantify the drug associated with lipoprotein, it is essential to determine the protein concentrations of the lipoproteins. In all drug loading experiments, the anthracycline concentration was found to be >0.10 g/L (11) and as high as 0.29 g/L (12). In these cases, the ratio of Dox to protein was high enough (for example, 25 μg drug/mg of protein) to interfere in the color development of protein in the BCA method by >50%. The protein concentration measured by the BCA method would be 1 g/L with 25 μg of Dox/mg of protein when the actual protein concentration was <0.5 g/L. The magnitude of interference is higher when the drug-to-protein ratio is higher (Fig. 1 ). Protein concentration is used as a bench mark to quantify cells and to express activity of an enzyme. In many cytotoxicity and enzymatic studies, the Dox concentration was found to be significantly high (7 μg to 0.29 mg of Dox/mg of protein) (12). The results from our studies indicate a significant increase in the color development of the BCA reagent with Dox in a concentration-dependent manner in the BCA method but not in the Bradford assay. School of Pharmacy, Memorial Univ. of Newfoundland, St. John’s, NF A1B 3V6, Canada Dox interference in the BCA protein assay. Aliquots of 20 μL of sample containing 0.25, 0.50, 1.0, 2.5, 5.0 × 10−1 g/L of each of the following were assayed: Dox (□), BSA protein calibrator (○), and BSA calibrator containing Dox (BSA:Dox = 1:1) (▵). The data points are the mean ± SD of quadruplicate determinations (where vertical lines are missing the SD was less than the size of the symbol). This study was supported in part by a research grant to H.L. by the Banting Research Foundation.