ANALYTICAL

204,

BIOCHEMISTRY

107-109

(19%‘)

Coomassie Blue Protein Dye-Binding Assays Measure Formation of an Insoluble Protein-Dye Complex Thomas

Marshall

and Katherine

M. Williams

Biochemistry Research Laboratory, School of Pharmaceutical & Chemical Galen Building, Green Terrace, Sunderland SR2 7EE, Great Britain

Received

December

Polytechnic,

17, 1991

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 proteinCBB 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 !PI 1992 Academic Press, Inc. structure.

Coomassie blue protein dye-binding assays exploit the interaction between protein and Coomassie brilliant blue (CBB)’ G-250, solubilized in ethanol/phosphoric acid (1) or perchloric acid (a), for total protein assay. The Bradford option (1) is commonly used although its mechanism is poorly understood. Binding probably involves van der Waals forces and hydrophobic interactions between the anionic form of the dye and basic (Arg, His, Lys)/aromatic (Phe, Tyr, Trp) amino acid residues on the protein (3). Electrostatic interactions between protonated amino groups (protein) and sulfonic acid groups (dye) may also contribute (4). Protein assay involves monitoring an increase in absorbance at 595 (1) or 620 nm (2) within 1 h of adding the dye reagent. Longer time intervals result in precipitation of the protein-dye complex but it is commonly believed that the initial complex is soluble (1,2). Our studies indicate that centrifugation or filtration of the initial assay mixture results in complete loss of color yield. This indi’ Abbreviations

Sciences, Sunderland

used:

CBB,

Coomassie

0003-2697192 $5.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form

brilliant

blue.

cates that the colored protein-CBB dye complex is insoluble and suggests that solubility may dictate the varying response of the assay to different proteins and its requirement for macromolecular structure. MATERIALS

AND

METHODS

Test Proteins Bovine albumin (Cat. No. A-4503, A-7638), human serum albumin (A-8763), a-casein (C-7891), a-chymotrypsin (C-4129), cytochrome c (C-2506), hemoglobin (H-2500), lysozyme (L-2879), ovalbumin (A-5503), pepsin (P-6887, P-7012), and transferrin (T-4515) were purchased from Sigma Chemical Co. Ltd. (Poole, UK). Protein solutions were prepared gravimetrically (1 mg/ ml) in phosphate-buffered saline (2 g/liter Na,HPO,, 0.6 g/liter KH,PO,, 7.0 g/liter sodium chloride), 0.15 mol/liter sodium chloride or deionized water (MilliQ Water Purification System, Millipore, UK). All protein solutions were freshly prepared and centrifuged (12,000 rpm, 5 min; MSE Micro Centaur centrifuge) prior to use. Assay Procedures Bradford assay. Dye reagent was freshly prepared as recommended (1) by dissolving 0.1 g of Serva blue G (or Sigma brilliant blue G) in 50 ml of 95% ethanol, adding 100 ml of 85% (w/v) phosphoric acid, diluting to 1 liter with water, and filtering immediately prior to use. For assay, duplicate 25-100 pg amounts of protein (1 mg/ ml) in an assay volume of 100 ~1 was mixed with 5 ml of added dye reagent and the increase in absorbance (595 nm) measured, against a reagent blank, using a Jenway 6100 spectrophotometer (Dunmow, UK). The proteindye mixture was immediately centrifuged (12,000 rpm, 5 min; MSE Micro Centaur centrifuge) or filtered (0.2 pm; 107

Inc. reserved.

108

MARSHALL

-0.4

1 25

I ;“g

FIG. 1. Color bovine albumin

I 75

AND

I 100

10

40 $BSA

yield response of the Bradford (A) and Sedmak and Grossberg standard protein before (0) or after (0) centrifugation.

Sedmak and Grossberg assay. Dye reagent was freshly prepared as recommended (2) by dissolving 0.6 g of Serva blue G in 1 liter of 3% perchloric acid and filtering immediately prior to use. For assay, duplicate lo-40 pg amounts of protein (1 mg/ml) in an assay volume of 0.5 ml were mixed with 0.5 ml of dye reagent and the increase in absorbance (620 nm) measured against a reagent blank. The protein-dye mixture was immediately centrifuged or filtered, and absorbance measurement repeated on the respective supernates and filtrates.

AND

-0.4-

BSA

Minisart filter unit, Sartorius, UK) and absorbance measurement repeated on the respective supernates and filtrates.

RESULTS

WILLIAMS

(B) CBB

protein

dye-binding

assays

to increasing

amounts

centrate to the recovered supernate generated the expected color yield of the standard assay procedure. This indicates that protein is soluble in the ethanol/phosphoric acid. Thus, the binding of CBB to the protein must cause the precipitation. This may provide nucleation sites for further deposition of the dye such that the solubility properties of the protein in the dye reagent may dictate the response of the assay and its requirement for macromolecular structure. In the context of the present study bovine albumin, ovalbumin, and pepsin were simultaneously assayed us-

1.6

DISCUSSION

A comparison of the color yield response of the Bradford and Sedmak and Grossberg CBB protein dye-binding assays to increasing protein amount is shown in Fig. 1. Both assays show complete loss of color yield in the respective supernates following centrifugation (Fig. 1) or filtration (results not shown). The centrifuged supernates gave negative absorbance values relative to the centrifuged reagent blank and became more negative with increasing protein amount. This is consistent with the loss of reagent dye in the precipitated protein-dye complex. Precipitation of the protein-CBB dye complex was confirmed by the Bradford assay of a range of additional proteins (Fig. 2). In each case, immediate centrifugation of the assay mixture resulted in complete loss of color yield. This occurred irrespective of whether the protein was dissolved in buffer, saline or water, or the dye reagent prepared with Serva blue G or Sigma brilliant blue G. A mixture of protein solution with reagent devoid of dye followed by centrifugation and addition of dye con-

of

after

I 25

I 50

I 75

I 100

,ug PROTEIN FIG. 2. Color yield response of the Bradford assay to increasing amounts of hemoglobin (o), cytochrome c (O), transferrin (A), wcasein (0), ovalbumin (V), and pepsin (0) before or after centrifugation. Lysozyme and n-chymotrypsin gave an identical response to transferrin and ovalbumin, respectively.

COOMASSIE

BLUE

ASSAYS

MEASURE

ing the biuret (5), Lowry (6), or bicinchoninic acid assays (7). In each case, centrifugation of the assay mixtures resulted in no loss of color yield in the recovered supernates.

COMPLEX

3. Compton,

109

FORMATION

S. J., and Jones,

C. G.

(1985) Anal. Biochem. 151,369-

374. 4. Fazekas de St. Groth, S., Webster, R. G., and Datyner, Biochim. Biophys. Acta 71,377-391. 5. Gornall,

A. G., Bardawill,

C. J., and

David,

M. M.

(1949)

A. (1963) J.

Biol.

Chem. 177,751-766. 6. Lowry,

REFERENCES 1. Bradfbrd, 2. Sedmak,

544-552.

M. M. (1976) J. J., and

Anal. Biochen.

Grossberg,

72, 248-254. Anal. Biochem. 79,

S. E. (1977)

0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (19511 J. Biol. Chem. 193, 2655275. 7. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, E. M., Olsen, B. J., and Klenk, D. C. (1985) Anal. Biochem. 150,76-85.