An evaluation of protein assays for quantitative determination of drugs

We have evaluated the response of six protein assays [the biuret, Lowry, bicinchoninic acid (BCA), Coomassie Brilliant Blue (CBB), Pyrogallol Red-Molybdate (PRM), and benzethonium chloride (BEC)] to 21 pharmaceutical drugs. The drugs evaluated were analgesics (acetaminophen, aspirin, codeine, methadone, morphine and pethidine), antibiotics (amoxicillin, ampicillin, gentamicin, neomycin, penicillin G and vancomycin), antipsychotics (chlorpromazine, fluphenazine, prochlorperazine, promazine and thioridazine) and water-soluble vitamins (ascorbic acid, niacinamide, pantothenic acid and pyridoxine). The biuret, Lowry and BCA assays responded strongly to most of the drugs tested. The PRM assay gave a sensitive response to the aminoglycoside antibiotics (gentamicin and neomycin) and the antipsychotic drugs. In contrast, the CBB assay showed little response to the aminoglycosides and gave a relatively poor response with the antipsychotics. The BEC assay did not respond significantly to the drugs tested. The response of the protein assays to the drugs was further evaluated by investigating the linearity of the response and the combined response of drug plus protein. The results are discussed with reference to drug interference in protein assays and the development of new methods for the quantification of drugs in protein-free solution.     

Interference of N-hydroxysuccinimide with bicinchoninic acid protein assay

We report here substantial interference from N-hydroxysuccinimide (NHS) in the bicinchoninic acid (BCA) protein assay. NHS is one of the most commonly used crosslinking agents in bioanalytical sciences, which can lead to serious potential errors in the BCA protein assay based protein estimation if it is present in the protein analyte solution. It was identified to be a reducing substance, which interferes with the BCA protein assay by reducing Cu²⁺ in the BCA working reagent. The absorbance peak and absorbance signal of NHS were very similar to those of bovine serum albumin (BSA), thereby indicating a similar BCA reaction mechanism for NHS and protein. However, the combined absorbance of NHS and BSA was not additive. The time–response measurements of the BCA protein assay showed consistent single-phase kinetics for NHS and gradually decreasing kinetics for BSA. The error in protein estimation due to the presence of NHS was counteracted effectively by plotting additional BCA standard curve for BSA with a fixed concentration of NHS. The difference between the absorbance values of BSA and BSA with a fixed NHS concentration provided the absorbance contributed by NHS, which was then subtracted from the total absorbance of analyte sample to determine the actual absorbance of protein in the analyte sample.     

Protein measurement using bicinchoninic acid: elimination of interfering substances

Protein quantitation based on bicinchoninic acid (BCA) is simple, sensitive, and tolerant to many detergents and substances known to interfere with the Lowry method. However, certain compounds often used during protein purification do interfere with the BCA protein assay. The response of the BCA chromophore to various interfering substances has provided insight into the mechanism of protein quantitation by BCA. Certain substances (e.g., glucose, mercaptoethanol, and dithiothreitol) elicit a strong absorbance at 562 nm when combined with the BCA working reagent. The absorbance appears to be identical to the normal response elicited by protein. Other agents (e.g., ammonium sulfate and certain ampholytes) diminish the protein-induced color development and shift the wave-length of the color response. Both types of interference can be eliminated by selectively precipitating protein with deoxycholate and trichloroacetic acid (A. Bensadoun and D. Weinstein (1976) Anal. Biochem. 70,241-250) prior to reaction with bicinchoninic acid. The modifications described here permit quick, efficient removal of many interfering substances that are commonly utilized during protein purification.     

Evaluating the compatibility of three colorimetric protein assays for two-dimensional electrophoresis experiments

To evaluate compatibility of commonly used colorimetric protein assays for 2-DE experiments, we investigated the interfering mechanisms of major 2-DE component(s) in the Lowry-based assay, the Bradford assay and the bicinchoninic acid (BCA) assay. It was found that some 2-DE components did not directly interfere with the assays' color development reaction, but possibly influenced the quantitation results by interacting with proteins. Generally, simultaneous presence of 2-DE components in the samples demonstrated a cooperative rather than additive interference. Interference by reductants in the Lowry-based assay and the BCA assay were too prominent and could not be completely eliminated by either the reported alkylation procedure or the water dilution procedure. The Bradford assay however, presented a more suitable method for quantitating 2-DE samples because it was less interfered by most 2-DE components. Furthermore, despite slightly compromising protein solubility, utilization of reductant free 2-DE sample buffers conferred application of the Lowry-based and BCA assays in the 2-DE experiments.     

Interference of biogenic amines with the measurement of proteins using bicinchoninic acid

The use of bicinchoninic acid (BCA) to measure protein concentrations has received wide acceptance because the reagent is insensitive to many of the buffers, sucrose solutions and detergents used with various tissue and enzyme preparations. However, any compound capable of reducing Cu2+ in an alkaline medium such as biogenic amines will produce a color reaction. The primary objective of this study was to determine whether biogenic amines present in neuronal tissue would interfere with the measurement of protein using the BCA method. Catecholamines were found to produce a linear increase in color of the BCA reagent at concentrations between 1 and 100 nmol/2.1 ml assay volume. Catecholamines appeared to be more sensitive to the BCA reagent than either serotonin or ascorbic acid. Catecholamines at concentrations of 50 nmol/mg of protein or 1 nmol/2.1 ml assay volume or higher will produce significantly (P less than 0.0001) higher color reactions than protein alone. The BCA reagent is not ideal for measuring protein concentrations of intact synaptic vesicles and chromaffin granules since the catecholamine concentrations in these organelles are high enough to increase the color developed by 1.1 to 2.5 times that observed with protein alone. The linearity of the color development produced by catecholamines suggest that BCA could be used to quantitate catecholamine concentrations between 1 and 100 nmol. The BCA reagent will not distinguish between the different catecholamines.     

考马斯亮蓝显色液组分对蛋白质测定的影响

考马斯亮蓝（CBB）显色法测定蛋白质含量的主要缺点之一是线性关系差．通过研究显色液组分对线性关系的影响,发现显色液H⁺浓度是影响线性关系的主要因素,并提出了一个新的显色液配方来改善考马斯亮蓝蛋白质测定法的线性关系．     

Coomassie blue protein dye-binding assays measure formation of an insoluble protein-dye complex.

The solubility of the protein-Coomassie brilliant blue (CBB) complex formed upon Bradford (Anal. Biochem. 72, 248-254, 1976) or Sedmak and Grossberg (Anal. Biochem. 79, 544-552, 1977) protein assay has been investigated by centrifugation or filtration of the assay mix within 10 min of adding dye reagent. The results show complete loss of color yield in the respective supernates and filtrates. This indicates that the protein-CBB complexes are insoluble at the time of absorbance measurement. Protein solubility in the dye reagent may dictate the relative response of the assay to an individual protein and the requirement for macromolecular structure.     

Evaluation of colorimetric assays for analyzing reductively methylated proteins: Biases and mechanistic insights

Colorimetric protein assays, such as the Coomassie blue G-250 dye-binding (Bradford) and bicinchoninic acid (BCA) assays, are commonly used to quantify protein concentration. The accuracy of these assays depends on the amino acid composition. Because of the extensive use of reductive methylation in the study of proteins and the importance of biological methylation, it is necessary to evaluate the impact of lysyl methylation on the Bradford and BCA assays. Unmodified and reductively methylated proteins were analyzed using the absorbance at 280 nm to standardize the concentrations. Using model compounds, we demonstrate that the dimethylation of lysyl ε-amines does not affect the proteins' molar extinction coefficients at 280 nm. For the Bradford assay, the responses (absorbance per unit concentration) of the unmodified and reductively methylated proteins were similar, with a slight decrease in the response upon methylation. For the BCA assay, the responses of the reductively methylated proteins were consistently higher, overestimating the concentrations of the methylated proteins. The enhanced color formation in the BCA assay may be due to the lower acid dissociation constants of the lysyl ε-dimethylamines compared with the unmodified ε-amine, favoring Cu(II) binding in biuret-like complexes. The implications for the analysis of biologically methylated samples are discussed.     

pH and buffering in the bicinchoninic acid (4,4'-dicarboxy-2,2'-biquinoline) protein assay

The HCO3/CO3(2-) buffer used in the bicinchoninic acid (BCA) protein assay has only weak buffering capacity at the recommended pH (11.25). Consequently the assay is rather sensitive to interference from effectively acid or alkaline samples, particularly in the micro method. Adjustment of pH in these alkaline solutions of high [Na+] is complicated by Na+ errors on the pH electrode. Hence it is recommended to prepare the buffers from known amounts of NaHCO3 and Na2CO3, and to reduce the pH to around 10.7; this offers much better buffering capacity with only a limited reduction in color development.     

Sulfo-N-hydroxysuccinimide interferes with bicinchoninic acid protein assay

This study revealed a major interference from sulfo-N-hydroxysuccinimide (sulfo-NHS) in the bicinchoninic acid (BCA) protein assay. Sulfo-NHS, a common reagent used in bioconjugation and analytical biochemistry, exhibited absorbance signals and absorbance peaks at 562 nm, comparable to bovine serum albumin (BSA). However, the combined absorbance of sulfo-NHS and BSA was not strictly additive. The sulfo-NHS interference was suggested to be caused by the reduction of Cu²⁺ in the BCA Kit’s reagent B (4% cupric sulfate) in a manner similar to that of the protein.     

The highly abundant urinary metabolite urobilin interferes with the bicinchoninic acid assay

Estimation of total protein concentration is an essential step in any protein- or peptide-centric analysis pipeline. This study demonstrates that urobilin, a breakdown product of heme and a major constituent of urine, interferes considerably with the bicinchoninic acid (BCA) assay. This interference is probably due to the propensity of urobilin to reduce cupric ions (Cu²⁺) to cuprous ions (Cu¹⁺), thus mimicking the reduction of copper by proteins, which the assay was designed to do. In addition, it is demonstrated that the Bradford assay is more resistant to the influence of urobilin and other small molecules. As such, urobilin has a strong confounding effect on the estimate of total protein concentrations obtained by BCA assay and thus this assay should not be used for urinary protein quantification. It is recommended that the Bradford assay be used instead.     

Quantitative immunoblot assay for assessment of liposomal antibody conjugation efficiency.

Routine direct assessment of immunoglobulin (Ig)-liposome(lp) conjugation efficiency has been impeded by phospholipid interference with standard protein and immunoassay methods. Rabbit IgG conjugated to anionic liposomes was quantitated in immunoblots using computer image analysis techniques. Lp-coupled Ig was separated from free Ig by dialysis in disposable Spectra/Por units (MWCO 300 kDa). Differential Lowry protein assay (DLA) of the thiolated Ig reactant and the dialyzate provided an estimate of conjugation efficiency that was compared to the results of the immunoblot assay (IBA). The color response of Ig-lp in the IBA was about an order of magnitude greater than rabbit IgG alone, requiring the synthesis of an Ig-lp standard in which the Ig conjugation efficiency was assessed by radiotracer methodology. The use of the same standard in three colorimetric protein assays verified the accuracy of the IBA and demonstrated that the colorimetric assays could be employed to determine Ig-lp conjugation efficiency. In terms of sensitivity and specificity, however, the IBA is better suited for routine assessment of laboratory-scale Ig-lp conjugation efficiencies. The DLA was found to be an unsatisfactory measure of conjugation efficiencies because an interfering substance was apparently released by Ig-lp preparations.     

Protein analysis of mammalian cells in monolayer culture using the bicinchoninic assay

We have applied the bicinchoninic acid (BCA) protein assay to rat brain primary astrocyte monolayer cultures growing in multiwell culture plates. The BCA method provides a more rapid and sensitive procedure with greater stability of color than is obtained using the Lowry method. Also, large numbers of samples can be read rapidly at the available wavelengths on an enzyme-linked immunosorbent assay microtiter plate reader. We found, however, artifactually high readings when using isotonic buffered sucrose to wash the cultures followed by sodium hydroxide to solubilize the cell protein. Such a procedure is commonly used for washing monolayer cell cultures in transport and binding studies. This effect was found to be due to hydrolysis of sucrose to the reducing sugar glucose. Use of Triton X-100 eliminated this problem, but this agent only solubilized about 80% of the protein that could be solubilized with sodium hydroxide. Furthermore, the high viscosity of Triton X-100 makes it more difficult to use. We found that washing the cells with isotonic mannitol solution followed by solubilization with sodium hydroxide gave reliable results. The sensitivity and speed of this method makes it suitable for multiple protein determinations in experiments using large numbers of cell culture samples.     

Protein concentration of cerebrospinal fluid by precipitation with Pyrogallol Red prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis

The Pyrogallol Red Molybdate (PRM) and Coomassie Brilliant Blue (CBB) protein dye-binding assays have been applied to samples of cerebrospinal fluid (CSF) to investigate protein concentration by dye precipitation prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration values of the CSF samples (N=62) showed good agreement between the PRM and CBB assays as indicated by linear regression analysis (y(PRM)=1.033x(CBB)+1.004 in units of mg/l, r=0.99) but the PRM assay was optimal for protein concentration as the PRM protein-dye complex was less soluble allowing protein recovery over a wider working range. Dye precipitation using PRM is recommended as a simple, rapid and economic method for protein concentration of samples of CSF prior to SDS-PAGE.     

Dynamics of a partially stretched protein molecule studied using an atomic force microscope

The dynamics of a single protein molecule subjected to forced mechanical unfolding was investigated in a millisecond time domain using a custom-made atomic force microscope (AFM) apparatus, which allows simultaneous measurements of an average tensile force applied to a single molecule and its mechanical response with respect to an external oscillation. Our target protein was genetically engineered bovine carbonic anhydrase II (BCA) which is a monomeric globular protein, and it has been shown that the as-expressed BCA from Escherichia coli contains two conformational isomers, one with enzymatic activity (type I) and the other without (type II). An interesting feature observed from the dynamic measurements was that when the type I BCA conformer was extended, it often exhibited a clear out-of-phase response against an external oscillation. The type II BCA conformer, however, always exhibited an in-phase response to the external oscillation. This relationship between different types of BCA and their dynamical behaviors was evidently observed around the discontinuous transition point from type I to II.     

Differential effects of chlorpromazine on secretion, protein phosphorylation and phosphoinositide metabolism in stimulated platelets.

Increasing concentrations of chlorpromazine (30-500 microM) caused a progressive lysis of gel-filtered platelets, as monitored by the extracellular appearance of cytoplasmic ([14C]adenine-labelled) adenine nucleotides. The chlorpromazine-induced lysis was markedly enhanced by thrombin and phorbol ester, and complete cytolysis was found at chlorpromazine concentrations of 100 microM and above in the presence of thrombin. At non-lytic concentrations, chlorpromazine caused a dramatic increase in the thrombin- or phorbol ester-mediated incorporation of 32P into phosphatidylinositol 4-phosphate and, to a lesser extent, into phosphatidylinositol 4,5-bisphosphate in platelets pulse-labelled with [32P]Pi. Chlorpromazine alone also caused an incorporation of 32P into the phosphoinositides. Non-lytic concentrations of chlorpromazine had no effect on the phosphorylation of the 47 kDa protein (regarded as the substrate for protein kinase C), but markedly inhibited the accompanying secretion of ATP + ADP and beta-hexosaminidase when platelets were incubated with 0.17 microM-phorbol ester or 0.1-0.2 unit of thrombin/ml. At lower concentrations of thrombin, chlorpromazine did not inhibit, but slightly enhanced, secretion. A protein of 82 kDa was phosphorylated during the interaction of platelets with thrombin and phorbol ester, and this phosphorylation was enhanced by chlorpromazine (non-lytic). These results suggest that the previously reported inhibition of protein kinase C by chlorpromazine is probably non-specific and due to cytolysis. However, since non-lytic concentrations of chlorpromazine inhibit secretion, but not protein kinase C, in platelets, activation of protein kinase C is not involved in the stimulation-secretion coupling, or chlorpromazine acts at a step after kinase activation. Possible mechanisms of this inhibition by chlorpromazine are discussed in the light of its effect on phosphoinositide metabolism and protein phosphorylation.     

Modification of the bicinchoninic acid protein assay to eliminate lipid interference in determining lipoprotein protein content.

The bicinchoninic acid (BCA) assay method for the determination of protein has been investigated for its utility in measuring the protein content of plasma lipoproteins. Although other methods, principally those based on the method of Lowry et al. (1951, J. Biol. Chem. 193, 265-275) have been extensively used for this purpose, the tolerance of the BCA method to many commonly encountered detergents and buffers offers a definite advantage over the Lowry-based methods. In this study, lipoprotein protein values obtained by the BCA method were compared to a standard modification of the Lowry et al. procedure since this assay forms the basis of much of the relevant literature. The standard BCA assay was found to overestimate the protein content of very low density lipoprotein by approximately 70% and low density lipoprotein by approximately 30%; high density lipoprotein values compared favorably. Overestimations by the BCA assay paralleled the relative phospholipid content of the lipoprotein fractions. This apparent lipid effect was eliminated by the addition of 2% sodium dodecyl sulfate to samples prior to the analysis. In the presence of this detergent, BCA assay measurements for these three lipoprotein fractions were 97, 90, and 98%, respectively, of the reference assay values.     

Interferences of suspended clay fraction in protein quantitation by several determination methods

Seven current methods of protein quantitation, Bradford (standard, micro, and 590/450 nm ratio), Lowry, bicinchoninic acid (BCA), UV spectrophotometry at 280 nm, and Quant-iT fluorescence-based determination, were compared with regard to their susceptibility to interferences due to the presence of suspended and not easily detectable clay particles. Bovine serum albumin (BSA) and Na-Wyoming montmorillonite were selected as model protein and reference clay, respectively. Protein-clay suspension mixtures were freshly prepared for each assay to simulate supernatants not completely centrifuged in batch sorption/kinetic experiments. Seven fixed increasing levels of clay (0.0, 0.00725, 0.0145, 0.029, 0.058, 0.145, 0.435 mg ml⁻¹) were mixed with different levels of BSA in an appropriate range for each assay. To ascertain the interfering effect of different levels of clay, the theoretical concentrations of BSA were plotted against the estimated BSA concentrations of the samples, as obtained from the calibration curve of each method. A correct quantitation of the BSA concentration not influenced by clay would be described by a regression line with slope (b) not significantly different from 1 and an intercept (a) not significantly different from zero. At the lowest clay levels (0.00725 mg ml⁻¹) a significant interference was evident for Bradford micro, Bradford 590/450, UV, and fluorescence. The three methods (Bradford standard, Lowry, and BCA) that seemed to show the better performances in the presence of clay after this first screening step also underwent an ANCOVA analysis, with the measured BSA concentrations as dependent variable and the clay concentrations as covariate. The Bradford standard and BCA methods were affected by a clay-dependent interference on BSA quantitation. The Lowry assay was the only method that gave correct estimates of BSA concentrations in the presence of any of the clay levels tested.     

Serum protein and enzyme levels in rats following administration of antioxidant vitamins during caffeinated and non-caffeinated paracetamol induced hepatotoxicity.

The effects of caffeinated and non-caffeinated paracetamol administration, with or without vitamins A and E supplementation on the protein and enzyme levels in Wistar albino rats were investigated using cafeinated paracetamol and paracetamol as caffeinated and non-caffeinated paracetamol respectively, and water soluble acetic acid derivatives of vitamins A and E. Serum AST, ALT and ALP levels (u/l) significantly increased [P < 0.05] following paracetamol administration. Caffeination as well as administration of vitamins A and E caused significant decreases[P < 0.05] in AST and ALP levels in all test groups when co-administered with paracetamol and in ALT level except in the cafeinated paracetamol + Vitamin E group in which ALT and ALP level except in the cafeinated paracetamol + vitamin E group in which ALT and ALP levels significantly increased [P < 0.05]. Total serum protein level (g/100ml) significantly increased following caffeination as well as during co-administration of cafeinated paracetamol and Vitamin E; and significantly decreased during co-administration of paracetamol and vitamin A. Paracetamol administration without caffeination or supplementation with vitamin A and E can therefore cause increases in serum liver enzymes that is suggestive of liver necrosis which can be ameliorated to varying degrees by caffeine, vitamin A and E.     

Protein determination using bicinchoninic acid in the presence of sulfhydryl reagents

The bicinchoninic acid (BCA) copper reagent, developed for quantification of proteins, was found to react with thiol reagents in a linear and reproducible manner. The reactivity with thiols closely matched the extinction coefficient determined for the Cu(I)-BCA complex [6.6 X 10(3) liters (mol Cu.cm)-1], suggesting that the reaction is quantitative. This reaction interferes with the accurate determination of protein concentrations. A method was developed for determining protein concentrations in the presence of thiol reagents using the BCA protein reagent. The procedure involves preincubation of the protein solution with iodoacetamide prior to addition of the BCA protein reagent. Iodoacetamide does not react with the BCA reagent by itself. In the presence of a 10-fold molar excess of iodoacetamide over thiol equivalents, the reaction of the thiol with the BCA reagent is prevented. The method is simple and allows the assay of solutions of proteins which have been stabilized by the addition of thiol reagents.