How to Illustrate Ligand-Protein Binding in a Class Experiment An Elementary Fluorescent Assay Alain Marty', Mkhel Boiret, and Michel Deumle Universite de Perpignan, Laboratoire de Chimie Physique, Avenue de Vilieneuve, 66025 Perpignan Cedex, France An important function of proteins that is of prime interest in chemistry, biology, and pharmaceutical or medical sciences is the fixation of drugs and their transport. In order to illustrate the hinding of a small molecule to a protein, we propose the following fluorescent assay to students of a Chemistry and Biology S e c t i ~ n By . ~ using an appropriate fluorescent ligand and a given protein we apply the fluorescent probe technique to measure the number of binding sites, the number of site classes (a class includes many equivalent sites), and their association constants ( I ) . This spectroscopic method has been selected because fluorescence is a sensitive and convenient method widely used by chemists and biologists for multiple purposes. In comparison with the equilibrium dialysis method, which is time consuming, our fluorimetric approach can he achieved in five hours. Reagents
8-Anilino-l-naphtalenesulfonicacid (ANS) and bovine serum alhumine crystalline (BSA) were obtained from Sigma Chemical Company. All experiments were performed in phosphate huffer, pH 7.4, with I = 0.1. The other solvents used were tetrahydrofuran (THF) of spectroscopic grade from Merck and deionized water. Fluorescence spectra were recorded a t 25+2OC on a Perkin-Elmer LS3 fluorescence spectrophotometer without spectra corrections. A molecular weight of 69,000 was used for BSA preparations, and the albumin concentrations were determined assuming a molar extinction coefficient of 46,000 cm-' mol-' l a t 278 nm(2).
Fisure 1.Fluorescence spema of A N S (I) in pureTHF (3X lo@mol I-'): (2)in buffer OH = 7.4 14.4 X mol 1-9 in THF-water mixtures as exDressed bv Volum; percentages: la) 9416. ID) 89/11, tcl80120. la1 67/33. (e1'50/50.A I sol.toans showed an absoroance of 0.30 a1 360 nm Excitstlon wavelength: 360 nm.
Experiments ANS Fluorescence in Aqueous and Nonaqueous Solutions ANS fluoresces weakly in pure water, hut its emission is drastically enhanced in a nonaqueous medium like THF. Figure 1shows the fluorescence intensities of ANS depending on the composition of the water-THF mixture used as solvent. In addition to the observed change in quantum yields, a red shift of the emission maximum results from increase of the water content in the solvent mixture. Because the ANS fluorescence is much larger in a nonaqueous medium than in water, this molecule can he usedfor probing the BSA hydrophobic sites (3). ANS-BSA Binding Studies In a solution of ANS (1.62 X mol I-') in huffer phosphate were added successive volumes of a BSA solution (1.40 X mol I-'). Corresponding fluorescence intensities were measured simultaneously. As the fluorescence quantum yield (6)of the bound ANS is much larger than the corresponding @ of the free ANS, the total fluorescence increases (Fig. 2). When all the ANS molecules contained in the medi-
' Author to whom correspondence should be addressed.
Third year of "License de Chimie et Biologie," Universite de Perpignan.
Figure 2. Variations of Me fluorescenceintensity of an ANS solution (1.62 X 10V mol I-') as a function of BSA concentrations.
um are hound to the protein sites a limit of the fluorescence is reached. I-'), the For agivenconcentration of BSA (2.13 X 10-"01 fluorescence titration was carried out for several ANS concentra~ions.Figure 3 shows that similarly the tluorescence intensirv increases untilall the HSAsitesareoccupied by the ligand. Dlscusslon The hinding of ANS to BSA sites was examined by following the equilibrium L+S+LS
where L, S, and LS represent, respectively, the free ligand, Volume 63 Number 4
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Figure 3. Fluwometric titration of 50 ml of aBSA solution (2.13 X 10-'mol I-') by a solution of ANS (9.69 X 1 0 P ma1 I-'). ANS volumes added were.
.
Figure 4. Scatchard plot of the binding of ANS l o BSA: experimental; ~~k"1ated.
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respectively, (1) 0.6 mi, (2) 1.2 ml. (3) 2.0 ml, (4) 3.1 ml. (5) 5.5 ml. Curve 6 corresponds toan ANS saiutlon (9.69 X 10@ mol I-', gain X 10) w i W u l BSA.
the vacant sites, and the occupied sites of the protein. If K is the equilibrium constant, [BSAIt the total protein concentration in solution, n the number of equivalent binding sites, and r the number of moles of bound ligand per mole of protein, we have K = r ([L](n- d)-'
found for all points along the titration rurve. The resultsare lotr red in Fiaure 4. The curvatureofthe Scatchard d o t thus bhtained indicates that ANS binds to more than onk class of sites ( 5 )and that the simple form of the Sratchard equation (eq 1) is no longer valid. when two classes of protein binding sites are assumed, eq 2 is used (5)
with r = [LS] [BSA],-I
Such a protein equilibrium is usually evaluated by the Scatchard equation
Using the approximation method of Cantor and Schimmel (6) the data noints fitted hv such a nonlinear function eives the following four parametkrs. n, = 1.05
This equation allows a quantitative interpretation of the ANS-BSA hinding. In experimental conditions, only the hound ANS fluoresces, and the fluorescence limit IF,(see Fig. 2) is obtained when ANS molecules are fully bound. Besides, IF is proportional to lLSl (i.e.. to hound ANS) in the ranee of concentrations used ( 4 j . ~o&equently,the'fluorescence intensity was used for determinine the concentration of the occupied sites. I.et us show u,ith the results of curve 2 (Fig. 3) the use of the fluorescence intensitv rmeasured at 470 nm) tocnlculate = 13.0 in [LS], r, [L], and r [ ~ ] - l . - ~ i g u r2e shows that arbitrary units when [LS] = 1.62 X 10-6 mol I-'. Therefore, when the fluorescence intensity is 16.9, [LS] = 2.106 X mol I-'. Then, from initial concentrations [Lit = 2.27 X lo-" mol I-' and [BSAIt = 2.08 X 10-"01 I-', we get [L] = 1.64 X 10-7 mol I-', r = 1.01, and r [L]-I = 6.17 X lo6 mol 1-I. Similarly, values for the fraction of hound probe were
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Journal of Chemical Education
Kt = 1.75 X
lo7 mol-' 1
The eood anreement achieved hetween calculated and experim&tal data (Fig. 4) demonstrates that in our experimental conditions two classes of hinding sites may be proposed as reported previously by Jun et al. (7). The former class has onlv one bindine site with a hieh association constant K1 whiie the latter hgs two bindingsites characterized by a lower association constant Kz. Literature Cited 11) SudiowG.; Birkett D. J.; Wade N. D. Mol. Pharmol. 1975,Il. 824. I21 Takenake 0.;Nishimura Y.:Takcnaka A.; Shibata K. Biochim. Biaphya. Acto 1970.
"",.""".
I61 Cantor. C. R.:Schimmel, P. R. In "Biophysical Chemistry"; New York. 1971:pt 3, p 256.
(7) Jun. H. W.; Luzzi, L. A,; M a , J K. J. Phorm. Sci. 1915,64,49S.
