ANALYTICAL

195,14-17

BIOCHEMISTRY

(1991)

Interference of Biogenic Amines with the Measurement of Proteins Using Bicinchoninic Acid’ Tracy

L. Slocum

Department

Received

and Jean

of Pharmacology,

October

D. Deupree’

University

of Nebraska

Medical

Press,

68198-6260

10, 1990

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 nmol12.1 ml assay volume. Catecholamines appeared to be more sensitive to the BCA reagent than either serotonin or ascorbic acid. Catecholamines at concentrations of 50 nmollmg of protein or 1 nmoh2.1 ml assay volume or higher will produce significantly (P < 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. o IBSI Academic

Center, 600 South 42nd Street, Omaha, Nebraska

Inc.

Measurement of protein concentration in various biological systems has always been impeded by the presence of reagents that also react with reagents used in the measurement of proteins. A recent method developed ’ This research was supported by a grant (NS 15187) from the National Institutes of Neurological and Communicative Disorders and Strokes. * To whom correspondence should be addressed.

by Smith et al. (1) is based on the reduction of Cu2+ to Cu+ by protein. The Cu+ then complexes with bicinchoninic acid (BCA)3 (Pierce BCA protein reagent), which absorbs light at 562 nm. Although this method appears to be insensitive to most buffers, sucrose and detergents, anything that can reduce Cu2+ to Cu+, will produce the characteristic purple color associated with the binding of Cu+ with bicinchoninic acid. It is not surprising that biogenic amines, which are easily oxidized in alkaline media in the presence of metal ions, would produce the necessary reduction of Cu2+ for the formation of Cu+-bicinchoninic acid complex. The primary objective of this paper was to determine whether the concentration of biogenic amines present in various tissues from brain or the adrenal medulla might interfere with the measurement of protein concentrations in these tissues using the BCA assay. To answer this question we examined the sensitivity and specificity of BCA for catecholamines. The results indicate a significant increase (P c 0.0001) in the color development of the BCA reagent for catecholamines at concentrations in the assay that are 1 nmol or greater. This would be equivalent to a catecholamine concentrations of 50 nmol/mg protein or greater. The results also indicate that BCA assay could be used to measure catecholamines concentrations which are in the range of 1 to 100 nmoU2.1 ml assay volume. However, this assay lacks the ability to distinguish between the different catecholamines.

MATERIALS

AND

METHODS

BCA assay. The BCA reagent was purchased from Pierce (Rockford, IL, 6105) and used according to the instructions supplied with the reagent. Samples of either biogenic amines in 0.1 mM HCl or 0.02 to 0.1 mg protein in water were added in a volume of 0.1 ml. BCA 3 Abbreviation

used:

BCA,

bicinchoninic

acid.

14

0003-2697/91$3.00

Copyright All

rights

0 1991 by Academic

of reproduction

in any

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Press, Inc. reserved.

BIOGENIC

AMINES

AND

BICINCHONINIC

15

ACID

25,

I

-

i

z6

a k

,.--

5

NE

. .

1.0

0.0 0.5

WI.

i

,.,,.,,,,,.,,1,,.,

0

5

10

Biogenic Amines (0.05 Irmole)

TIIIE

FIG. 1. Reaction of biogenic amines with BCA. Catechol, dopamine (DA), norepinephrine (NE), epinephrine (EPI), tyrosine (TYR), serotonin (5HT), tryptophan (Try), and ascorbic acid at a concentration of 0.05 wmoUO.1 ml were incubated for 2 h at room temperature with 2.0 ml of the BCA protein reagent (Pierce). The absorbance of the samples at 562 nm was determined. The data are presented as the mean of triplicate determinations f SE.

reagent (2 ml) was added. Color was allowed to develop for either 2 h at 25°C or 30 min at 37”C, as indicated. Statistical analysis. Statistical analysis was conducted using the Student’s t test. RESULTS

AND

DISCUSSION

The reaction of 50 nmol of various biogenic amines with the BCA protein reagent at room temperature for 2 h indicated that a wide range of biogenic amines will react with the BCA reagent to produce the characteris-

FIG. 3. nephrine. per 0.1 ml measured

15

20

(hr)

Rate of increase in color absorbance produced by norepiThe color produced by incubation of 25 (0) and 50 nmol (A) of norepinephrine with 2.0 ml of BCA reagent at 25°C was after 0.33, 0.67, 1, 1.5, 2, 2.5,3,4, and 19 h.

tic purple color (Fig. 1). Dopamine was the most sensitive of the catecholamines (Fig. 1). There was no significant difference between the absorbance due to norepinephrine or epinephrine. Serotonin was less sensitive to the BCA reagent than the catecholamines but more sensitive than catechol. Ascorbic acid, which is present in most tissue samples containing biogenic amines, as well as the amino acids, tyrosine and tryptophan, also produced the color change with the BCA reagent. Tests to determine the sensitivity of the BCA reagent to catecholamines indicated that changes in absorbance

,-

0 20

40

60

Cotecholamlne

60 [nmole] i

0.11 0

-11

-10 Log Cotacholomina

-9

-8

-7

1

2 Norepinephrine

3

4

5

(nmol)

[mole]

FIG. 2. Sensitivity of catecholamines to color development with the BCA reagent. Norepinephrine (0, l ), epinephrine (V, v), and dopamine (m) were reacted with 2.0 ml of the BCA protein reagent at concentrations between 1 pmol and 100 nmol10.1 ml for either 2 h at room temperature (@, v) or 30 min at 37°C (0, V). Data points represent the mean of triplicate determinations + SE.

FIG. 4. Color development produced by norepinephrine in the presence of bovine serum albumin. The color produced by 0.75 to 5 nmol/ 0.1 ml of norepinephrine reacting with 2.0 ml of BCA reagent in the presence of 20 pg of protein was measured after 2 h incubation at 25°C. The data points are the mean + SE of quadruplicate determinations. *P < 0.02, **P < 0.001 compared to assays without norepinephrine.

16

SLOCUM

AND

DEUPREE

A

0.00

0.02

0.04 m

FIG. 5. Color development added to various aliquots and quadruplicate determinations. (M) nmol of norepinephrine. norepinephrine per milligram

0.06

0.06

0.10

(md

0.00

0.02

0.04 6s

0.06

0.08

0.10

h)

produced by bovine serum albumin in the presence and the absence of norepinephrine. The BCA reagent was the color allowed to develop over a 30 min time period at 37°C. The data points represent the mean +- SE of A. Color development of various concentrations of bovine serum albumin in the presence of 0 (O), 5 (A), and 10 B. Color development of aliquots of bovine serum albumin containing 0 (O), 20 (A), and 600 nmol (a) of of protein.

were detectable at concentrations in the assay media between 1 and 100 nmol12.1 ml assay volume (Fig. 2). Dopamine produced slightly higher absorbance values than either norepinephrine or epinephrine. There appeared to be linear increase in absorbance between 1 and 100 nmol of catecholamine suggesting that the BCA method could be used to quantitate catecholamines concentrations that are in the 10 to 1000 PM range (1 to 100 nmoV2.1 ml assay volume). The development of color associated with the reaction of protein with the BCA reagent is slow at room temperature and enhanced by increasing the temperature (1). Color development with 20 pg of protein for 2 h at room temperature is equivalent to that developed over 30 min at 37°C and less than that produced after 15 min at 60°C (1). Color changes produced by the reaction of protein with the BCA reagents continues to develop, at a slow rate, over the next 24 h when left at room temperature (1). We therefore wanted to know the time required for color development associated with catecholamine reaction with the BCA reagent at room temperature and at 37°C. At room temperature the color development produced by catecholamines changes rapidly over the first 1.5 h (Fig. 3). There is only a 15% further increase in color over the next 17 h. The color development after 2 h at room temperature is similar to that developed over 30 min at 37°C (Fig. 2). Are the tissue biogenic amine concentrations high enough to interfere with the measurement of protein concentrations at the cellular or subcellular level? When assaying tissue samples that contain catecholamines, the significance of the effects of the catecholamine on color development will depend on the concen-

tration of catecholamine and the ratio of catecholamine to protein. The absorbance change produced by 1 nmol of norepinephrine reacting with BCA reagent for 2 h is 0.03 (Figs. 2 and 4). This is significantly (P < 0.0001) different from that produced without norepinephrine. Bovine serum albumin, at concentrations between 0.02 and 0.1 mg did not appear to interfere with the color developed by 5 and 10 nmol of norepinephrine (Fig. 5A). These results suggest that catecholamine concentrations of 5 nmoV0.1 mg protein or higher will produce color intensities that are significantly (P -K 0.001) higher than those due to the protein alone. Large dense cored vesicles from splenic nerve contain between 3 and 70 nmol/mg protein (2). Intact chromaffin granules have catecholamine concentrations in the range of 600 nmol/ mg protein (3). The color absorbance due to the catecholamines in intact chromaffin granules is 2.5 times that produced by the protein alone (Fig. 5B). Chromaffin granules that have been lysed to reduce their catecholamine content still contain a residual amount of catecholamine at a concentration of 20 nmol/mg protein (3). This concentration does not appear to significantly alter the color intensity produced by bovine serum albumin (Fig. 5B). The catecholamine content of brain tissue samples ranges from 6 pmol/mg to 0.7 nmol/mg protein (4,5), and should not interfere with protein determinations using the BCA reagent. The Coomassie blue method for measuring protein developed by Bradford (6) and modified by Spector (7) does not react with 50 pmol of catechol, dopamine, epinephrine, norepinephrine, serotonin, tyrosine, tryptophan, or ascorbic acid (data not shown). It is possible, therefore, to use the Coomassie blue method for mea-

BIOGENIC

AMINES

AND

suring protein concentrations under conditions where the catecholamine concentrations are above 50 nmol/ mg protein. Pollard et al. (8) have demonstrated identical results for protein quantitation of adrenal gland subcellular fractions using the Coomassie blue method and the Lowry (9) after removing the catecholamines. These results indicate that catecholamines will significantly alter color development produced by proteins at catecholamine concentrations that exceed 50 nmol/mg protein. Serotonin and ascorbic acid will also influence color development with the BCA reagents, but to a lesser extent than catecholamines. These studies also demonstrate the feasibility of using the BCA reagent to quantitate biogenic amines at concentrations that are in the 10 PM to 1 mM range using a O.l-ml sample volume. This would be an alternative to the spectroflurometric or electrochemical detector methods currently used for measuring concentrations of biogenic amines that have been separated using a HPLC system.

BICINCHONINIC

17

ACID

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2. Klein,

R. L., Lagercrantz, H., and Zimmermann, transmitter Vesicles, p. 135, Academic Press,

3. Deupree, J. D., Weaver, J. A., and Downs, Biophys.Acta 714,471-476.

4. Schneider, (1984)

M. B., Murrin, Clin. Neuropharm.

L. C., Pfeiffer, 7,247-257.

A. K., N. M.,

H. (1982) New York.

D. A. (1982)

NeuroBiochim.

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J. D.

5. Palkovits, M., and Brownstein, M. J. (1989) in Handbook of Experimental Pharmacology (Trendelenburg, U., and Weiner, N., Eds.), Vol SO/II, pp. 8-11 Springer-Verlag, New York. 6. Bradford, M. M. (1976) Anal. B&hem. 72,248-254. 7. Spector,

T. (1978)

Ad.

Biochem.

86,142-146.

8. Pollard, H. B., Menard, R., Brandt, H. A., Pazoles, C. J., Creutz, C. E., and Ramu, A. (1978) Anal. Biochem. 86, 761-763. 9. Lowry, (1951)J.

0. H., Rosebrough, N. H., Farr, Biol. Chem. 193,265-276.

A. L., and Randall,

R. J.