The Binding of Coomassie Brilliant Blue A Physical Biochemistry Experiment J. L. Sohl and A. G. Spliigerber Gustavus Adolphus College, St. Peter, MN 56082 The reversible binding of small molecules or ligands to ororein molecules is important because such interactions are very common in living systems. Receptors located on the surfaces of cell membranes often consist of embedded proteins that small such as hormones. For this ..---~ - bind ~ ~ molecules ~ reason somk' uhdetstanding of the nature of binding interactions is considered of sufficient importance to be included in undergraduate biochemistry courses (1-3). In the biochemical laboratory macromolecule-ligand binding is studied by a variety of techniques in which the concentration of either free or bound macromolecules or ligand may be monitored. The most often mentioned method of study is the equilibrium dialysis technique, which makes use of radioactively tagged ligands and a ligand-permeable membrane ( 4 4 ) . However, for a variety of reasons equilibrium dialysis is not suitable as an undergraduate hiochemistry laboratory exercise. A more convenient method than equilibrium dialvsis is a howspectrophotometric one if i t is practicable. In ever.. s~ectroohotometrv is often not possible because the . spectral characteristics neither mac~omoleculenor ligand change to an avpreciable extent when ligand binding occurs. ~ b i s - ~ a ~d eekri b e s a system in which a large change in molar absorptivity of the ligand, the dye Coomassie Blue (CBB), occurs when i t binds to a protein molecule such as Bovine Serum Albumin (BSA). The suitability of the CBB-BSA system as an experimental model for protein-ligand binding is discussed and the determination of exoerimental h a n d binding curves as a biophysical chemistry laboratory experiment isdescrihed. A linear form of a binding equation is derived, and experimental BSA-CBB bindingdata are treated t o determine numbers of binding sites on the protein as well as values for intrinsic binding constants.
Materials and Methods Coomassie brilliant blue (CBB) G-250 was a product of EastmanKodak Co. (Rochester,NY). BSA was obtained from Sigma Chemical Co. (St. Louis. MO). Other chemicals wer.i~ur~hasedfrom Fisher chemical Co. (~ittihurgb,PA). The spectral data were generated with a single beam Beekman DU monochromator equipped with a Gilford electronics system for digital readout of absorbances. Any spectraphotometerhaving good baseline stability with an absorbance range from 0 to 1and readability to 0.001 absorbance units would be suitable. For rhr determination of bind~ngdata, the dye reagent formulation of I'eterson ,141 was usrd,ar folltrws:0.92Jgof CRBdye powder was added t o 1W ml. 95% ethanol and 2UU ml. of 8% nhosphoric acid, and the resulting mixture was diluted to 1200 mi. he dye reagent solution was mixed thoroughly, filtered through Whatman No. 1paper, and stored in an amber bottle. In order to determine points on a CBB-BSA binding curve, constant volumes of dye reagent were placed in aseries of 13-X 100-mm test tubes, and the solution volume in each tube brought to 2 mL with the ethanol-phosphoric acid-water dye solvent. Variahle amounts (from 0 to 1.0 mL) of a 10 mg/mL stock BSA solution were then added to each tube. and the total volume in each tube adiusted to mixtures ..2.0 . . mL with deionized water. The resultine ..dvenrotein . . were mned thoroughly hy stirring,transferred in sequence tua l-rm Pyrex cuwtre. and the ahsorbancea measured st 595 nm against a hlank of deionized water. Although theahaorbancechangemayhemeasuredat a number of different wawlenshs 1101,in the binding experiment derailed here. 595 nm is used beeause the laxest absorbance change occurs at this wavelength, and the most accurate binding data are therehy obtained.
The Dye-Protein System The interaction of the Coomassie Blue dye molecule (structure shown in Fig. 1)with proteins is the basis of one of the newer methods of protein assay (7)that has gained in popularity in recent years (8). The protein assay method makes use of an acidic dye reagent in which the pH of an aqueous CBB solution is adjusted to a value below one by addition of phosphoric acid. The dye molecule is thereby converted by protonation to a reddish colored species that can bind to most if not all protein molecules (9).When this red species binds to protein, the bound dye molecule convertsio a blue specie< and the resulting color change may be measured spectropbotometrically. The change in absorhanee that a~small amount of vrotein is added to ~ -o&rs ~when ~ the acidic dye reagent is usually monitoied a t 595 nm, although large absorbance changes occur at other wavelengths as well (10). A major advantage of the CBB system as a method for protein assay is its extreme sensitivity, less than 5 pg of protein being easily detected. The major disadvantages,
Figure 1. Struchrre d Coomassle Brilliant Blue. 0 2 5 0
~
of
~~~~~
~
262
~
which are shared by traditional protein assays such as the Biuret (11)and Folin-Ciocalteau (12)methods, include variability of color response from one protein to another and nonlinearity of the assay curve (absorbance versus mass of protein present) (13).
~
~~~
~
~~~
~~~
~
~
Journal of Chemical Education
In order that accurate binding data may be found, scrupulous attention to detail must be paid. Accurate adjustable digital pipets are best. The deliverv of constant re~roducibleamounts of dve reagent and accurate amounts of stock protein solution to each t&e is essential. Thorough rinsing of the cuvette with deionized water followedby careful drying with Laboratory tissue is required. Mixing of the dye-protein solutions should be done in such a way that formation of froth is minimized. Vortexing is not recommended. Frequent zeroing of the instrument with deionized water is also necessary, depending on the amount of instrument drift. Experimental Blndlng Curves
Experimental binding curves are generated at constanr dye levels and upwardly varying protein concentration. Figure :!shows a set of four binding curves determined at four constant dye levels. These curves show that at sufficiently hieh orotein levels all dve molecules become bound to nrot e h is indicated by the plateau in absorbance values. Binding curves are more eenerallv olotted in terms of Y. the fractronal saturation ojligand binding sites, or 0,tbd average number of lieands bound oer macromolecule. Where the b&dingstudiesaiecarried ouiat constant dye levelsas is done here, it is convenient to let Y equal the fraction of dve molecules converted to the bound foim. The expression for Y then becomes: A
sites with the same intrinsic binding constant seems realistic if, as has been proposed (16),the primary dye-bihding sites on the protein consist of oiie type or one class of amino acid side chain. It is also realistic that the sites be independent because cooperativity between sites would not be expected in an artificial binding situation. The Binding Equation In order to derive the binding equation, consider a protein P with three equivalent and independent binding sites for the dye D. Each site bas intrinsic binding (association) constant k. Using standard statistical factors in the usual manner (17), one has K, = 3k, K2 = k, and K3 = kl3, where the K's are macroscopic constants. Equilibria and binding constant expressions are given by P+D=PD;
K, = (PD)I(P)(D); (PD) = 3k(P)(D)
PD
+ D = PD,;
(2)
K, = (PDJ/(PD)(D); (PDJ = 3k2(P)(D)' (3)
Total dye concentration DT is given by: D, = (Dl
where Abs,,,l. is the absorbance of a particular protein-dye solution, Abs,,, is the maximum absorbance observed when sufficient protein is added so that all dye molecules are converted to the orotein-bound form. and Absn is the zero protein absorbance. Using the Y notation, bindkg curves of Y versus total protein concentration may be easily constructed. These are qualitatively very similar to the curves of Figure 2. Inorder to treat thedata most efficiently, it is desirahle to derive an equation for the bindinpr curve ( Y versus PT in this case, where-& is total protein c¢ration). In favorable cases the equation may be linearized and straight line plots such as Scatchard plots (15) may be drawn. If a plot of the binding data confirm to this linear equation, then it may be number of oossible to calculate bindine constants andlor ~~, .binding sites from slopes anlintercepts. The most realistic model for the Dresent case assumes all binding sites to be equivalent and independent. Equivalent
= (Dl = (Dl
+ (PD) + 2(PD,) + 3(PD,) + 3k(P)(D) + 6kZ(P)(D)'+ 3k3(P)(D)3 + 3k(P)(D)(l+ k(D)I2
Total protein concentration PTis given by:
Rearranging,
The amount of bound dye is given by:
~~
by substitution for (P) from eq 7. Division by DT yields the binding equation:
This result may beeasily extended toany number of binding sites. The general binding equation for any numher of binding sites n is: Y=
nk(D)(P,lD,) 1 k(D)
+
A linear form of this equation is found by taking reciprocals of both sides of eq 10 and multiplying by PT:
Using the fact (see eq 9)that (D)/DT = 1- Y,
8
12 MG. BSA
16
:
Figwe 2. Plots of absorbance at 595 nm Venus mass of BSA 0ontaI"ed In a total volume of 3.0 mL at four constant CBB levels. A, 0.125 mL dye reagent; A, 0.25 mL dye reagem; 0 , 0 3 7 5 mLdye reagent; E. 0.50 mLdye reagem.
Equation 12 is one of a t least three linear forms of eq 10. This form allows the most useful plots of the binding data. If the data yield a linear plot, then in all probability only one class or type of binding site is involved in dye binding, Volume 68
Number 3
March 1991
263
stant) on the BSA molecule. On the basis of studies carried out on the interaction of CBB with polyamino acids (161, the strong color response with polymers of the basic amino acids histidine, lysine, and especially arginine indicates the positively charged side chains of these amino acids probably constitute the primary dye binding sites on protein molecules. The five orimarv bindine sites indicated bv the binding data may tierefore he thenumber of basic amino acid side chains heine in the -proper - environment for dve bindineto occur. From the same study (16) on the interactions of polyamino acids with CBB, a weaker response was noted for the aromatic amino acids tyrosine, tryptophan, and phenylalanine. I t has been suggested (18) that a spatial juxtaposition of a basic amino acid side chain and the side chain of an aromatic amino acid mav be reouired for initial dve bindine. In this case, the five primary binding sites may be the number of hasic-aromatic amino acid oairs occurrine in the BSA molecule. The approximately 25 secondary binding sites may be interpreted on the basis of studies on the amount of CBB taken up during staining of protein bands in polyacrylamide gels (19). From these studies i t appears that dye molecules mav also bond to dve molecules already bound to protein, creiting a "stacking" interaction between the phenil rings of adjacent dye molecules. In view of this possibility, short chains of up to five stacked dye molecules would be areasonable explanation of the number of secondary binding sites. Accordine to the above interoretation of the bindine events, the iarger intrinsic binding constant of 9.2 X lo4M-7 would represent an ionic interaction between the ~ositivelv basic &no acid side chains and negatively charged sulfo". nate mourn .. . on the dve molecule. The smaller intrinsic binding constant of 4.2 ~ 1 0W' ' would then represent a hydrophobic or van der Waals interaction between . phensl - rings of adjacent dye molecules.
-
Figure 3. BSA-CBB blcding data plotted a c c w d i n g t o equation 12. Cons~tant CEB level of 0.20 mL dye reagent. Milliliters of s t o c k 10 mg1mL B S A used for
eachdatapoht 0.005, 0.01. 0.015. 0.020.0.025.0.030.0.040. 0.050.0.060. 0.070, 0.080. 0.10. 0.15, 0.20, 0.25.0.30, 0.35, 0.40, 0.45, 0.50.
and the number of such sites and the intrinsic bindine constant may be found from the slope and intercept of the plot. If the plot turns out not to be linear, then the most probahle explanation is that there is more than one class of dye hinding site on the protein with different intrinsic hinding con-
~ e s u n and s Dlscusslon
Figure 3 shows binding data for BSA plotted according to eq 12. In order t o construct the plot, PT for each solution studied is calculated from the gram amount of protein Conclusion present in a 3-mL volume using a molecular weight for BSA This paper presents a laboratory experiment involving the of 68,000 daltons. The plot is seen t o have the appearance of binding of a ligand, Coomassie Blue, to a macromolecule, two intersecting straight lines, which would be expected if Bovine Serum Albumin. The success of the method depends the protein had two classes of binding sites with quite differon the spectral ~rooertiesof the CBB molecule. which ent intrinsic binding constants. change markedly k h l n binding occurs. Although the'macroAt high protein levels (high values of PTIY and 1/(1- Y)) molecule-lieand svstem is auite artificial. and the bindine of one would exoect dve to be bound onlv to ~ r i m a r vbindine BSA could not be studied in the s h e natural ligands nites, those with thk largest intrinsic bind& las;ociation'i manner. the bindine studv presented here would nevertheconstants. The linear segment of the plot with smaller slope less give the ytudenrsome-idea as to how real systems might represents binding onlyio this class ofsite. Using a molecuhe aooroached and what data mieht be derived from such lar weight for the dye of 854 daltons and assuming pure dye, stud&. I t is hoped that this paperwill provide the basis fora a constant total dykconcentration (DT) value of 6 . 0 ~ o - ~ M useful experiment in an area that does not receive much is calculated for each solution studied. The intercept of the attention in the undergraduate biochemistry laboratory. plot then allows calculation of an n value of about 5 binding sites of the primary type. From the slope, the intrinsic binding constant is about 9.2 X lo4M-'. Lllerature Cited At low protein levels (low values of PT/Yand l ( ( 1 - Y)), 1. Mefder, 0.E. Bioehemialry: Academic: San Francisco. 1977: pp 187-200. where dve is in excess..i t would be expected that both orima2. Bohinski. R. C. Madern Concepts in Biochemistry. Sfhed.:AlIynand Bacon: Boston, ry&s and secondary sites having smaller intrinsic dinding 1987: PP 153-157. constants would be bound to dve. In this reeion the straieht 3. Stryer. L. Biochemisfgv, 3rd ed.: Freemen: New York. 1988: pp 154-155.239-240, 4. w n Holde, K. E. PhyeicoiBiochemi8lry. 2nd ed.; Prentiee-Hall: Englevoad Cliffs. NJ. line segment of the plot with larger slope represents binding 1986; PP 58-59. t o both orimarv and secondarv classes of bindine site. From 6. Bdl, J. E.: Bell, E. T. Protein3 and Enzymes; Prentice-Hall: Englovoal Cliff%.NJ, 1988: PP 379380. the inte;cept and slope of this-section of the plot; an n value 6. Dryer, R. L.; Lot& G. F. Expwimrnfol Biochemi$try: Oxford University: New York, of about 30 binding sites is found along with an average 1989:pp 103-104 7. Bradf0rd.M. M. Anal.Biochem. 1976,72,24S-254. intrinsic binding constant of 4.2 X lo3M-'for both classes of 8. Davis. E. M. Am. Biorrch.Lob. I988,6. 2&37. binding site. % Sedmak, J. J.;Cm&erg.S E A n o l . Riocham. 1977,79,514-552 I t should be noted that because the minimum value of the LO. Read. S. M.; Nartheote. 0 . H. A n d B i o c h a m . 1981,116.53-64. 11. Bai1ey.J. L. T~chniquesinProtrinChemisiry: Elsovier: NevYork, 1962;pp340-311. function 1/(1- Y) is 1, rather than 0, the Y intercept of the 12. Peferson,G. L.Ana1, Chrm. 1979,100,201-220. plot is found a t an x value of 1. If extrapolation to x = 0 is 13. Sp1lttgerber.A. G.: Snhl, J. Anal. Rbochem. 1989, 179. 196201. I d Peters0n.G. L.MrthodsEnlrm"l1981.91.9rlL9. carriedout, the linear section of the plot a t low protein levels 15. scatchard.G.Ann. N.Y. ~ ~Sci. ~1949.5j.660-872. d . has a negative intercept, rendering the data meaningless. 16. C0mpton.S. d.:.lones.C. G.Ano1. Riorhem. 1985, 751,389-374. r y ,ed.;Prentice-Hall: Ewlewood Cliffs, NJ, 17. van Hold*. K. E P h ~ ~ i c a l R ; o c h ~ m i r f2nd \Bith regard to interpreting the ralculated binding site "-, ec a" p ""*,. numbers, the value of approximately 5 would represent the 18. Tal, M.; Si1beniein.A.: Nu8ser.E.J. R i d Chem. 1985.260.99769980, 19. Wilron.C.Ano1. Biochem. 1979,96,263-278. number of strong binding sites (large intrinsic binding cou-
to
~. ~~
~~
~
Ln"*,
264
Journal of Chemical Education
