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The colored complex formed between Cu’ and bicinchoninic acid is the basis of the bicin-
choninic 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. B&hem. 150,76-85). Studies show that cysteine, cystine, 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°C indicate that tryptophan,
tyrosine, and the peptide bond are not completely oxidized at this temperature. When the
reaction temperature is increased to 6o”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, tyro-
sine, 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. o 198s
Academic F’res, Inc.
KEY WORDS: bicinchoninic acid: nrotein determination; spectrophotometry; amino acids;
interfering substances.
The use of bicinchoninic acid (BCA)’ to phan residues. The second source of Cu+ is
determine protein concentration (1) is rap- assumed to be a temperature-dependent reac-
idly gaining favor as an alternative to the tion of peptide bonds with Cu*+. This process
Lowry method (2) of protein determination is reported to be more prevalent as the incu-
(3-5). The sensitivity of the technique and its bation temperature is increased.
ease of use make the BCA assay preferable to In this paper, the results of experiments de-
the Lowry assay in many instances (1). The signed to investigate the sources of color for-
BCA assay has also been reported to be less mation in the BCA assay are described. Stud-
sensitive to interference ( 1) by a number of ies of the reaction between the BCA reagent
commonly encountered compounds. The as- and a number of compounds which might re-
say utilizes the color change resulting from sult in interference with the assay are also re-
the strong complex formed between Cu+ and ported.
BCA. Smith and coworkers (1) have postu-
lated that the Cu+ is produced by two sepa- MATERIALS AND METHODS
rate reactions. The first is temperature inde-
pendent and is postulated to arise from the The solutions from which the BCA work-
oxidation of cysteine, tyrosine, and trypto- ing reagent was prepared were obtained
from Pierce Chemical Co. Acetamidophenol,
’ Abbreviations used: BCA, bicinchoninic acid; BSA, ascorbic acid, biuret, cysteine, cystine, gluta-
bovine serum albumin; LSV, linear scan voltammetry; thione, N-acetyl-L-tryptophan, N-acetyl+
DPV, differential pulse voltammetry. tryptophanamide, and compounds 8, 14,25,
and 26 in Table 1 were supplied by Aldrich. trophotometer. A sample prepared using wa-
Bovine serum albumin (BSA), dithiothreitol, ter in place of the compound being tested was
3,4-dihydroxyphenylalanine, insulin, soy- used as the reagent blank. In studies designed
bean trypsin inhibitor, trypsinogen, trypto- to determine if the absorbances of the inter-
phan, and compounds 9, 10, 11, 12, 13, 18, fering compounds could be compensated for
23,24,27, and 28 (Table 1) were from Sigma. by a blank solution, a concentration of the
Tyrosine and compounds 15, 16, 17, 19, 20, interfering substance which was the same as
2 1, and 22 were obtained from U.S. Bio- that in the BSA sample was used as the re-
chemical, indole was supplied by MCB, and agent blank. A standard curve was obtained
gelatin was from J. T. Baker. The BCA work- using concentrations of BSA from 0.050 to
ing reagent was prepared by combining 50 1.O mg/ml in aqueous solution using a water
parts Reagent A (an aqueous solution of 1% blank. The apparent concentration of the
Na,BCA, 2% Na2C03. H20, 0.16% Naz tar- BSA in the presence of each interfering sub-
trate, 0.4% NaOH, and 0.95% NaHC03 at pH stance was determined from the standard
11.25) with 1 part Reagent B (4% Cu- curve after subtraction of the absorbance of
SO,. 5H20) and mixing (1). Solutions of the the blank. The Lowry assay was carried out
20 commonly occurring amino acids and the according to published procedures (2).
compounds listed in Table 1 were prepared at Linear scan voltammetry (LSV), differen-
a concentration of 1 mM. Compounds which tial pulse voltammetry (DPV), and con-
were not soluble in water were dissolved in a trolled potential coulometry were performed
carbonate/bicarbonate buffer containing 2% with a Model 174a Polaragraphic Analyzer
Na2C03.H20 and 0.095% NaHC03 which (E, G and G, Princeton Applied Research)
had been adjusted to pH 11.25 with sodium coupled to an Omnigraphic 2000 xy recorder
hydroxide. Protein solutions were prepared (Houston Instruments) for LSV and DPV or
at concentrations of 0.050 to 1.0 mg/ml in
to an Omniscribe strip-chart recorder (Hous-
aqueous solution with the exception of insu- ton Instrument) for coulometry. A rotating
lin, which was dissolved in the pH 11.25 car-
glassy carbon electrode was the indicator
bonate/bicarbonate buffer. Solutions of the
electrode during LSV and DPV studies. The
interfering compounds were prepared at con-
electrode was prepared by cementing a cylin-
centrations which gave absorbances of less
drical piece of glassy carbon (Tokai Electrode
than 1.2. Concentration ranges used were
0.050 to 0.50 mM for ascorbic acid, 0.10 to Mfg. Co., Ltd.) into the end of a 6-mm-o.d.
0.50 mM for dithiothreitol, 0.02 1 to 0.26 mM glass tubing. The electrode was rotated with a
for 3,4-dihydroxyphenylalanine, 0.25 to 2.0 Sargent synchronous rotator at 30 Hz. Con-
mM for penicillamine, 0.20 to 2.0 mM for glu- trolled potential coulometry was performed
tathione, and 0.0 11 to 0.11 mM for acetami- at a platinum gauze working electrode. All re-
dophenol. ported potentials are relative to a saturated
Solutions containing the compound of in- calomel reference electrode. The electroana-
terest were reacted with the BCA working re- lytical studies were done in a three-compart-
agent using the standard protocol described ment water-jacketed cell at 26°C. In each case
by Smith and coworkers (I). The assay was the pH 11.25 carbonate/bicarbonate buffer
conducted by adding a 150~~1 aliquot of the was used as solvent. Prior to each electroana-
sample to 3 ml of the BCA protein working lytical study, the sample was deaerated with
reagent and incubating the resulting solution nitrogen that had been passed through a gas
at either 37 or 60°C for 30 min. After the sam- train consisting of two wash towers contain-
ple had been cooled to room temperature, its ing acidic ammonium metavanadate and
absorbance at 562 nm was determined using amalgamated zinc, and a final wash tower
a Perkin-Elmer Lambda 3B uv-visible spec- containing deionized water. During coulo-
COLOR FORMATION IN THE BICINCHONINIC ACID ASSAY 233
MOLARABSORFUVITIESOFSEVERALPOTENTIAL
metric studies the sample was stirred by bub- CU*+COMPLEXES
bling nitrogen through the solution.
Complex (562 A,, (nm) %lax
RESULTS AND DISCUSSION cl?+ 33.5 = a
Cu*+-tyrosine 78.2 605 89.5
The results of tests of the BCA reagent with Cu*+-tyrosine tartrate 18.8 645 31.0
a number of amino acids, amino acid deriva- Cu*+-tryptophan 30.7 * n
tives, and di-, tri-, and tetrapeptides are pre- Cu*+-tryptophan tattrate 22.8 647 31.3
sented in Table 1. Of the amino acids nor- Cu2+-cysteine 0 a a
mally found in proteins, the BCA reagent Cu*+-cysteine tartrate 0.9 = a
only reacts with cysteine, cystine, tyrosine, a The solution was turbid and no maximum was ob-
and tryptophan. The results in Table 1 indi- served although the turbid&dance due to scattering con-
cate that cysteine, cystine, tryptophan, and tinued to increase as the wavelength decreased.
234 WIECHELMAN, BRAUN, AND FITZPATRICK
TABLE 3
A SUMMARYOFTHERESULTSOFTHEELECTROANALYTICALSTUDIES
Note. The values reported represent the average of three to five measurements.
’ The wave/peak is on the background wave caused by solvent oxidation; consequently the results are inaccurate.
on the plateau of the anodic wave, and the teins with the BCA reagent for 30 min at 37°C
electrochemical oxidation was carried out to is shown in Fig. IA. The fact that gelatin,
completion. The results of the coulometric which contains less than 1 mol% of the color-
studies are also listed in Table 3. Uncertain-
ties listed in the table are standard deviations.
From the results it is apparent that the BCA
reagent responds to any oxidizable species
that can be more easily oxidized than the pH
11.25 buffer solution. A comparison ofthe re-
sults of this study with the absorbances ob-
served in the BCA and Lowry assays shows
that there is no correlation between redox po-
tential and color formation in either assay.
Consequently some other factor, perhaps
differences in the strength of the copper com-
plexes, must be responsible for the differences
observed in the two assays. It can be seen in
Table 3 that the value of the coulometric n is
I for cysteine and 2 for tyrosine and trypto-
phan. The fact that the absorbance produced
by cysteine is greater than that observed for
tyrosine or tryptophan (Table 1) supports the
observation that tyrosine and tryptophan are
not completely oxidized in the BCA assay at 0
either 37 or 60°C. 0.2 0.4 0.6 0.8 1.0