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.     

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.     

Measurement of protein using bicinchoninic acid

Bicinchoninic acid, sodium salt, is a stable, water-soluble compound capable of forming an intense purple complex with cuprous ion (Cu1+) in an alkaline environment. This reagent forms the basis of an analytical method capable of monitoring cuprous ion produced in the reaction of protein with alkaline Cu2+ (biuret reaction). The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concentrations. When compared to the method of Lowry et al., the results reported here demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, one-step analysis and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique.     

Quantitative benedict test using bicinchoninic acid

Cupric ion (Cu2+), in complex form, functions as a selective oxidizing agent for a variety of compounds in the qualitative Benedict test. Cupric ion is reduced to cuprous ion (Cu+) in the reaction. We found that the cuprous ion formed in this type of redox reaction could be detected and quantified using 2,2'-bicinchoninic acid. This reagent produced an intense purple complex with cuprous ion. The color development in this modified Benedict test was dependent on pH, temperature, and time. The reaction is insensitive to ethanol and sodium dodecyl sulfate. This improved method of the Benedict test has enhanced sensitivity and makes the quantitation of compounds possible. This method should be useful for studies of Benedict-positive compounds which are available only in small amounts.     

Measurement of protein using flow injection analysis with bicinchoninic acid

We have used the bicinchoninic acid reagent developed by Pierce Chemical Co. to measure proteins in a simple flow injection analyzer. The sensitivity is comparable to that of the Lowry method and no pipetting of reagents is needed. Results are obtained in less than 1 min and samples may be run at a rate of 60/h. The response is linear over a range of protein concentration (0-10 micrograms) and sample size (5-20 microliters) convenient for most analytical requirements. A peristaltic pump, a controlled-temperature water bath, and a spectrophotometer with flow cuvette are the only special apparatus required.     

Interference by lipids in the determination of protein using bicinchoninic acid

Bicinchoninic acid forms the basis of an analytical method for the determination of protein. The reagent produces a purple complex with cuprous ion (Cu+) in an alkaline environment and is the basis for the monitoring of cuprous ions produced in the reactions of proteins with alkaline Cu2+. This method of protein determination was reported to have greater tolerance to many commonly encountered interfering compounds, when compared to the Lowry technique. However, we have found the bicinchoninic acid technique to produce erroneously high values for protein when common membrane phospholipids were included in the assay. Phospholipids in the presence of bicinchoninic acid produced an absorbance peak similar to that produced by protein. This absorbance was linear with concentration, however, the slope varied for individual phospholipids. The combined absorption of phospholipid and protein was not strictly additive. The results indicate that the presence of appreciable quantities of lipid in samples can cause significant error in the analysis of protein by the bicinchoninic acid procedure.     

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.     

Determination of protein covalently bound to agarose supports using bicinchoninic acid

A method using bicinchoninic acid (BCA) for the direct determination of protein covalently bound to agarose is described. The method involves the preparation of a standard curve using solubilized protein, the determination of the slurry concentration of the gel sample, the colorimetric assay of the gel slurry using BCA in a serum separator device, and the calculation of the amount of protein bound to the gel using the standard curve of the solubilized protein. The use of the BCA method for direct estimation of immobilized protein agrees well with the "balance" method which depends upon the measurement of protein depletion and yields highly reproducible results with a variety of coupling chemistries commonly used with agarose beads. The use of commercially available serum filters provides for a fast, less than 1 h, and convenient analytical format.     

Spectrophotometric quantitation of anchorage-dependent cell numbers using the bicinchoninic acid protein assay reagent

A rapid and convenient method is described for the determination of the actual and relative number of adherent cells in tissue culture. The cell lines human melanoma C32, ATCC CRL 1585, mouse melanoma B16-F10, and pig epithelial LLC-PK1, suspended in Dulbecco's minimum essential medium containing no serum, were allowed to adhere to fibronectin adsorbed to wells of a 96-well microtiter plate. Nonadherent cells were removed by aspiration, wells were washed, and adherent cells were solubilized with 200 microliters of the bicinchoninic acid (4,4'-dicarboxy-2,2'-biquinoline) protein assay reagent. Plates were heated to 60 degrees C for 30 min and absorbances read at 562 nm using a microtiter plate reader. A linear correlation was observed between the number of adherent cells in the range 2-8 X 10(5)/ml cells added and the protein content of the adherent cells as measured by the BCA protein reagent. The assay procedure gave absorbance values in the range of 0.100 to 1.30 making the method highly sensitive and reproducible. Blank wells containing only coupled protein and no cells gave little or no absorbance. Cell adhesion was fibronectin specific since little or no cell attachment was observed when microtiter plates were coupled with bovine serum albumin. Similar results were obtained with other cell types such as platelets. These results indicate that measurement of total cellular protein using the BCA protein reagent can be a rapid and sensitive assay for the detection and quantitation of adherent cells.     

Comparative study of Lowry and Bradford methods: interfering substances

The methods of Lowry and Bradford, commonly used for protein determination, were compared regarding the level of interference of some substances used for glucoamylase precipitation by ethanol. The method of Bradford suffers no interference while the method of Lowry showed protein concentration values 20% increased in the presence of ethanol and Tris. Despite these interferences, the Lowry method can evaluate more accurately the increase of purity during fractionation, due to its sensitivity to low molecular weight (below 6 kDa) proteins and peptides.     

Estimation of peptide concentration by a modified bicinchoninic acid assay

Although biuret based protein assays are theoretically applicable to peptide measurement, there is a high level of interpeptide variation, determined largely by peptide hydrophobicity. This variation in peptide reactivity can be significantly reduced by heat-denaturation of peptides at 95 °C for 5 min in the presence of 0.1 M NaOH containing 1% (w/v) SDS, prior to incubation for 30 min at 37 °C in BCA standard working reagent. This modification to the standard bicinchoninic acid (BCA) assay protocol allows for an accurate, rapid, and economical estimation of the peptide concentration within an unknown sample.     

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.     

Amino acid analysis using phenylisothiocyanate prederivatization: elimination of the drying steps

Results of experiments on the procedure for amino acid analysis via analysis of the phenylthiocarbamyl amino acids are reported. It was found that yields of some amino acids varied in the presence of salt and with changes in the vacuum drying steps. An improved procedure is described which includes a standard addition of salt to the hydrolysate before drying it; the redrying step is omitted and the post derivatization drying is replaced by a simple addition of heptane to the reaction mixture.     

Quantification of protein in dilute and complex samples: modification of the bicinchoninic acid assay

The colorimetric assay using bicinchoninic acid (BCA) as test reagent is useful for quantitative protein determinations due to its high sensitivity, ease, and tolerance to various contaminations present in biological samples or added during purification. For removal of interfering substances, protein precipitations have been described. Yet, obstructions became apparent with diluted and complex samples. Therefore we tested different solvents for removal of such interfering contaminants from the protein precipitate, and 1 M HCl was identified as the useful washing agent. The protocol described allows simple and accurate microdetermination in microtiter plates of proteins from complex samples.     

Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation

The colored complex formed between Cu+ and bicinchoninic acid is the basis of the bicinchoninic acid protein assay (P. K. Smith, R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B.J. Olson, and D.C. Klenk (1985) Anal. Biochem. 150, 76-85). Studies show that cysteine, tryptophan, tyrosine, and the peptide bond are capable of reducing Cu2+ to Cu+. Electrochemical studies and the magnitude of the color changes observed when the reaction is carried out at 37 degrees C indicate that tryptophan, tyrosine, and the peptide bond are not completely oxidized at this temperature. When the reaction temperature is increased to 60 degrees C, significantly more color formation is observed for these three groups. Studies with di-, tri-, and tetrapeptides and with proteins indicate that the extent of color formation is not the sum of the contributions of the individual color producing functional groups. Compounds with functional groups similar to those of cysteine, cystine, tyrosine, or tryptophan are shown to react with the bicinchoninic acid reagent. The color formed by these compounds in the presence of bovine serum albumin cannot be compensated for by using a reagent blank containing an identical concentration of the interfering compound.     

The effect of interfering substances on the disinfection process: a mathematical model

AIMS: To gain a greater understanding of the effect of interfering substances on the efficacy of disinfection. METHODS AND RESULTS: Current kinetic disinfection models were augmented by a term designed to quantify the deleterious effect of soils such as milk on the disinfection process of suspended organisms. The model was based on the assumption that inactivation by added soil occurred at a much faster rate than microbial inactivation. The new model, the fat-soil model, was also able to quantify the effect of changing the initial inoculum size (1 x 10(7)-5 x 10(7) ml(-1) of Staphylococcus aureus) on the outcome of the suspension tests. Addition of catalase to the disinfection of Escherichia coli by hydrogen peroxide, resulted in changes to the shape of the log survivor/time plots. These changes were modelled on the basis of changing biocide concentration commensurate with microbial inactivation. CONCLUSIONS: The reduction in efficacy of a disinfectant in the presence of an interfering substance can be quantified through the use of adaptations to current disinfection models. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the effect of soil on disinfection efficacy allows us to understand the limitations of disinfectants and disinfection procedures. It also gives us a mechanism with which to investigate the soil tolerance of new biocides and formulations.