ANALYTICALBIOCHEMISTRY 175231-237 (1988)

Investigation of the Bicinchoninic Acid Protein Assay: Identification of the

Groups Responsible for Color Formation

KAREN J. WIECHELMAN, ROBERT D. BRAUN, AND JIMMIE D. FITZPATRICK

Department of Chemistry, University of Southwestern Louisiana, P.O. Box 44370, Lafayette, Louisiana 70504
Received March 16,1988

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
determine protein concentration (1) is rap-
idly gaining favor as an alternative to the
Lowry method (2) of protein determination
(3-5). The sensitivity of the technique and its
ease of use make the BCA assay preferable to
the Lowry assay in many instances (1). The
BCA assay has also been reported to be less
sensitive to interference ( 1) by a number of
commonly encountered compounds. The as-
say utilizes the color change resulting from
the strong complex formed between Cu+ and
BCA. Smith and coworkers (1) have postu-
lated that the Cu+ is produced by two sepa-
rate reactions. The first is temperature inde-
pendent and is postulated to arise from the
oxidation of cysteine, tyrosine, and trypto-
’ Abbreviations used: BCA, bicinchoninic acid; BSA,
bovine serum albumin; LSV, linear scan voltammetry;
DPV, differential pulse voltammetry.
phan residues. The second source of Cu+ is
assumed to be a temperature-dependent reac-
tion of peptide bonds with Cu*+. This process
is reported to be more prevalent as the incu-
bation temperature is increased.
In this paper, the results of experiments de-
signed to investigate the sources of color for-
mation in the BCA assay are described. Stud-
ies of the reaction between the BCA reagent
and a number of compounds which might re-
sult in interference with the assay are also re-
ported.
MATERIALS AND METHODS
The solutions from which the BCA work-
ing reagent was prepared were obtained
from Pierce Chemical Co. Acetamidophenol,
ascorbic acid, biuret, cysteine, cystine, gluta-
thione, N-acetyl-L-tryptophan, N-acetyl+
tryptophanamide, and compounds 8, 14,25,

231 0003-2697188 $3.00

Copyright 0 1988 by Academic Press, Inc.
All rights of reproduction in any form reserved.
232 WIECHELMAN, BRAUN, AND FITZPATRICK

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

TABLE 1 tyrosine give significantly different amounts

THEABSORBANCESPRODUCEDBYIIIIMSOLUTIONS of color formation at 37°C suggesting a par-
INTHE BCA ANDTHELOWRYASSAYS tial reaction of the BCA reagent with tyrosine
and tryptophan. The differences in the absor-
BCA’
bances of these two amino acids is much
Compound 37'c 6o'C Lowly b more dramatic than those observed with the
Lowry assay where absorbances of 1.043 and
I. Indole 0.322 0.621 0.564 0.947 were observed for 1 mM solutions of ty-
2. Tryptophan 0.774 1.589 0.947
3. Tyrosine 0.480 1.172 1.043 rosine and tryptophan, respectively (Table 1).
4. Cyst&e 1.180 1.357 Although cysteine and cystine show only lim-
5. Cystine 1.687 2.047
ited color formation in the Lowry assay (6)
6. N-Acetyltryptophan 0.477 1.060 0.407
7. N-Acetyltryptophanamide 0.547 1.122 0.653 they contribute significantly to the color for-
8. (Ala), 0.003 0.004 0.026 mation in the BCA assay.
9. Ala-Asp 0.022 0.025 0.032 Spectrophotometric studies of 3.2 mM
10. His-Gly 0.013 0.017 0.170
11. Wu)2 0.010 0.013 0.046 Cu2+ solutions in the pH 11.25 carbonate/bi-
12. Trp-Phe 0.256 0.785 0.772 carbonate buffer were performed in order to
13. Tyr-Phe 0.201 0.499 1.069 determine the amount of interference of sev-
14. Gly-Tyr 0.419 0.877 0.897
15. LW-Trp 0.230 0.684 0.56 1 eral Cu2+ complexes with the spectrophoto-
16. Leu-Tyr 0.342 0.763 0.957 metric assay. The molar absorptivity of each
17. Trp-Ala 0.217 0.656 0.766 solution was measured at the wavelength of
18. (Alah 0.157 0.541 0.613
19. (Glyh 0.092 0.391 0.580 the BCA assay (562 nm) and at the absorptive
20. Gly-Leu-Ala 0.215 0.613 0.653 maximum for the solution. Because tartrate
21. Val-Gly-Gly 0.120 0.483 0.648 is a component of the BCA reagent, studies
22. Gly-Gly-Tyr 0.602 1.351 1.358
23. Val-Tyr-Val 0.642 1.379 0.963 were performed both in the presence and in
24. Leu-Leu-Tyr 0.641 1.712 1.422 the absence of 0.16% tartrate. The results are
25. (Ala), 0.125 0.677 0.318 summarized in Table 2. It is evident from the
26. WY), 0.055 0.347 0.187
21. Phe-Gly-Gly-Phe 0.038 0.263 0.367
low molar absorptivities that Cu2+ solutions
28. Gly-Gly-Tyr-Am 0.591 1.352 1.295 containing the tested compounds do not sig-
nificantly interfere with the spectrophoto-
a Absorbances at 562 nm resulting from reaction with metric assays at 562 nm.
the BCA working reagent for 30 min at the indicated
temperature. Biuret and a number of dipeptides which
b Absorbances at 720 nm resulting from reaction with do not contain cysteine, cystine, tyrosine, or
the Lowry reagent.
TABLE 2

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,
tryptophan do not show appreciable color
formation with either the BCA or the Lowry
assays. It is interesting to note that the BCA
reagent did not react with alanyl-asparagine
or histidyl-glycine, which have been reported
to react to a limited extent in the Lowry assay
(6). Since Smith and coworkers (1) have pos-
tulated that the peptide bond contributes to
color formation, we also tested a number of
tri- and tetrapeptides which did not contain
any of the color-forming amino acids. The re-
sults in Table 1 indicate that all of the tri- and
tetrapeptides tested showed some color for-
mation at 37°C. The finding that tripeptides
give more color formation at this tempera-
ture than tetrapeptides is consistent with the
observation of Legler et al. (7) that (Ala), has
a higher molar extinction coefficient at 720
nm than (Ala), in its reaction with the Lowry
reagent. Legler and coworkers (7) postulate
that in order for oxidation of the peptide
bond to occur in the Lowry assay the Cu2+
ion must form a tetradentate complex with
groups on the peptide backbone.
The necessity of forming this complex ex-
plains the behavior of di-, tri-, and tetrapep-
tides in the Lowry assay. Dipeptides, which
cannot form the tetradentate complex, do not
react with the Lowry reagent. Tripeptides re-
portedly form more stable complexes with
Cu*+ than tetrapeptides due to the negative
charge on the carboxylate group which re-
places an uncharged oxygen in the complex
(7). The fact that the BCA reagent requires at
least a tripeptide in order to oxidize the pep-
tide backbone suggests that formation of a te-
tradentate Cu*+ complex is required for oxi-
dation of the peptide bond in the BCA assay.
The results in Table 1 also indicate that
different combinations of tri- and tetrapep-
tides produce different amounts of color for-
mation in the BCA assay. For example, at
37°C (Glyh produces an absorbance of 0.092
compared to a value of 0.2 15 for glycylleucy-
lalanine. Differences in the extent of color
formation were also observed when these tri-
and tetrapeptides were reacted with the
Lowry reagent (Table 1). In this case (Gly),
AND FITZPATRICK
had an absorbance of 0.580 compared to an
absorbance of 0.653 for glycylleucylalanine.
Differences in the extent of color formation
were also observed with both assays for tetra-
peptides containing different combinations
of amino acids.
All of the di-, tri-, or tetrapeptides contain-
ing tryptophan or tyrosine develop color
upon reacting with the BCA reagent; how-
ever, in all cases the absorbances are different
from those of the free amino acids (Table 1).
There are also significant differences in the
absorbances of each of the dipeptides con-
taining either tyrosine or tryptophan. For ex-
ample, at 37°C glycyltyrosine has an absor-
bance of 0.4 19 compared to a value of 0.20 1
for tyrosylphenylalanine and 0.480 for free
tyrosine. Although the differences observed
for tryptophan-containing dipeptides are less
dramatic, there is a larger difference between
free tryptophan (0.774) and the dipeptides
(e.g., 0.2 17 for tryptophanyl alanine). Similar
differences between the free amino acids and
the dipeptides were observed at 60°C. The
free amino acids and tyrosine- or tryptophan-
containing dipeptides also produced absor-
bances of different magnitudes in the Lowry
assay. Differences in the extent of color for-
mation were also observed for indole, trypto-
phan, N-acetyltryptophan, and N-acetyltryp-
tophanamide in both the BCA and the Lowry
assays (Table 1).
Since it has been reported that the amino
acid side chain has a significant effect on the
redox properties of indole (8,9), electrochem-
ical studies of some of the compounds to
which the BCA reagent responds were under-
taken to determine their redox characteris-
tics. None of the compounds exhibited ca-
thodic waves or peaks (corresponding to re-
ductions). The potentials at which the LSV
anodic waves and the DPV anodic peaks
were observed are listed in Table 3. Con-
trolled potential coulometry was used to as-
certain the number n of moles of electrons
transferred for each compound during the
oxidations. In each case the potential of the
working electrode was adjusted to a potential
 COLOR FORMATION IN THE BICINCHONINIC ACID ASSAY 235

TABLE 3
A SUMMARYOFTHERESULTSOFTHEELECTROANALYTICALSTUDIES

Concentration Average Average Coulometric

Compound range (mh4) 42 W) u-w Ep W) W-9 (4

Cysteine 1.18-6.61 0.398 f 0.064 0.404 f 0.038 1.01 kO.18

Tryptophan 1.15-4.13 0.205 + 0.025 0.282 f 0.007 1.99kO.17
Tyrosine 0.88-4.23 0.398 f 0.008 0.43 1 f 0.007 2.07 r 0.15
Indole 1.15-2.03 0.253 + 0.059 0.357 + 0.012 1.23 +0.17
GlY), 1.75-3.04 0.94” 0.813 +0.015” 0.8 rt 1.0”
WY), 1.34-2.62 0.766 + 0.020” 0.845 AZ0.035” 0.8 kO.5”
N-acetyltryptophan 0.52-5.32 0.217+0.19 0.289 + 0.013 1.96 z!z0.09

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

To investigate the effect of the temperature [Protein] (mglml)

dependent intensities we observed for tyro-
sine, tryptophan, and the peptide bond a
number of proteins with varying color-form-
ing amino acid side chains were utilized. A
plot of the absorbance at 562 nm produced
by reacting various concentrations of the pro-
FIG. 1. Standard curves resulting from the reaction of
varying concentrations of proteins with the BCA work-
ing reagent. (A) The reaction was carried out for 30 min
at 60°C. (B) The reaction was carried out for 30 min at
37°C. The proteins used were trypsinogen (A), insulin
(0), BSA (0), soybean trypsin inhibitor (Cl), and gelatin
(@I.
236 WIECHELMAN, BRAUN,
producing side chains (lo), gives significant
color formation at 37°C confirms our finding
that the peptide bond is oxidized in the BCA
assay at this temperature. The results shown
in Fig. 1A indicate that the total absorbances
observed for proteins deviate significantly
from the sum of the contributions of the indi-
vidual amino acids. For example trypsino-
gen, which contains 5.24 mol% cystine,
1.75% tryptophan, and 4.37% tyrosine and
has a total of 229 amino acids (1 l), produces
a significantly larger absorbance than BSA,
which contains 6.18% cysteine plus cystine,
0.35% tryptophan, 3.36% tyrosine and is
composed of 581 amino acids (11). This
finding is consistent with the results pre-
sented above which indicate that the absor-
bance produced by tyrosine or tryptophan in
di-, tri-, or tetrapeptides is different than that
observed for the free amino acid.
Although Smith and coworkers (1) re-
ported that at 60°C the difference in the ab-
sorbances of equal concentrations of gelatin
and BSA is significantly less than that ob-
served at 37”C, a thorough study of protein
to protein variation at the higher temperature
has not been reported. When the proteins are
incubated with the BCA reagent for 30 min
at 60°C the protein to protein deviations are
smaller than those observed at 37°C (Fig. 1B).
It is interesting to note that soybean trypsin
inhibitor, which contains 2.2% cystine, 1.1%
tryptophan, 2.2% tyrosine, and 181 amino
acids (12), produces a standard curve which
is lower in intensity than that obtained for
BSA at 37°C and greater in intensity than the
curve obtained for BSA at 60°C. This indi-
cates that again at this temperature the absor-
bances resulting from the reaction of the BCA
reagent with the protein of interest do not
represent a sum of the contributions of the
individual color forming groups.
In light of the ability of the BCA reagent
to oxidize cysteine, cystine, tryptophan, and
tyrosine, several compounds with similar
functional groups which might be present in
physiological samples were tested. Plots of
absorbance versus sample concentration for
AND FITZPATRICK
TABLE 4
THE AB~ORBANCES AT 562 nm RESULTING FROM THE
REACTION OF THE BCA WORKING REAGENT
WITH INTERFERING SUBSTANCES
Interfering
substance
Interfering plus 0.25
Compound substance” mp/ml BSAb
Acetamidophenol 2.42 0.799
Ascorbic acid 2.00 0.576
3+Dihydroxyphenylalanine 4.40 0.635
Dithiotlweitol 0.84 0.646
Glutathione 0.6 1 0.570
Penicillamine 0.41 0.515
’ The absorbance of 1 mM solutions calculated from
equations derived from linear regression. The samples
were reacted with the BCA working reagent for 30 min
at 37°C.
b The absorbances of 0.25 mg/ml BSA in the presence
of the interfering substances. The concentrations of the
interfering substances were 0.30 mM acetamidophenol,
0.18 mM ascorbic acid, 0.090 mM 3,4dihydroxyphenyl-
alanine, 0.45 mM dithiothreitol, 0.45 mM glutathione,
and 0.60 mM penicillamine. The reaction was ahowed to
proceed for 30 min at 37°C.
these compounds were linear in all cases. Lin-
ear regression analysis of the results gave cor-
relation coefficients greater than 0.9996.
When the equations derived from linear re-
gression were used to calculate the absor-
bances of 1 IIIM solutions of these com-
pounds, the results in Table 4 were obtained.
These results show that there is a considerable
variation in the sensitivity of the BCA reagent
for the compounds tested. The data in Table
4 suggest that the BCA assay is a relatively
nonspecific assay and that any compound
which can reduce Cu*+ to Cu+ provides a pos-
itive result. A series of experiments was car-
ried out to determine the effect of the interfer-
ing compounds on the accuracy of the BCA
assay. Solutions of BSA were prepared in var-
ious concentrations of each of the com-
pounds listed in Table 4. Concentrations
which produced absorbances in the range
0.23 to 0.56 were selected. The apparent con-
centration of the BSA was determined from
a standard curve using the absorbance of the
 COLOR FORMATION IN THE BICINCHONINIC
sample after subtraction of the absorbance
produced by the interfering compound.
When low concentrations of acetamidophe-
no1 (0.15 and 0.30 mM) and penicillamine
(0.12 mM) were used the apparent BSA con-
centrations were 100% of the actual concen-
trations. At higher concentrations of these
compounds and when ascorbic acid, dithio-
threitol, or glutathione was used as the inter-
fering compound the apparent BSA concen-
trations were as much as 25% less than the
actual concentrations. The fact that low con-
centrations of the interfering substances pro-
duce large absorbances upon reaction with
the BCA reagent (Table 4) and the observa-
tion that in many cases the absorbance of the
interfering substance cannot be adequately
compensated for using a reagent blank make
the BCA assay unsuitable with samples which
contain any of the compounds in Table
4. Consequently, although the BCA assay
is preferable to other commonly used pro-
tein assays in most cases, care must be exer-
cised when assaying samples which might
contain any of the compounds listed in Ta-
ble 4.
ACID ASSAY 237
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