The Modern Student laboratory Determination of Ligand-Macromolecule Binding Constants by a Competitive Spectrofluorometric Technique Caffeine Binding to Human Serum Albumin J. J. Inestal, F. GonzBlez-Velasco, and A. Ceballos
Departamento de Quimica Fisica, Facultad de Farmacia, Universidad de Salamanca, Apdo, 449, (37008) Salamanca The studv of the binding of small molecules (ligands) to . proteins is of great interest from different polnts of view, especiully in the field of biomediral sciences. Topics include dctailcd knou,ledg*:1. of the mechanisms ol'enzymatic nction and numerous intracellular reactions as well as distribution and hioavailahility of drugs in the human body. There are numerous techniques that enable the binding phenomenon to he quantified. Among these, those based on different types of spectroscopy have the advantage ofheing quite fast and simple. In particular, spectrofluorometric techniques have a series of characteristics that make them especially attractive. Spectrofluorometry is a very sensitive and selective teehnique: adequate selection of excitation and emission wavelengths allows us to analyze a particular substance in a relativelv. comdex . mixture. T h e spectral characteristics of the fluorescent emission are often greatly influenced by the characteristics of the surmundines of the molecule. Slight variations in the position of the energetic levels may modify the quantum 3eld of emission spectacularly because molecules whose fluorescence varies drastically with the polarity of the medium are relatively common. Moreover, the characteristicsof emission may provide information on the nature and properties of the hinding site ( I ) .
I n the relatively frequent case in which there are two types of equal and independent binding sites, the equation is as follows.
where the nlhinding sites have binding constant K I , and the n2 have hinding constant Kz. This equation cannot be linearized, and the values of the parameters nl, Kl, n2,and Kz must be obtained by numerical fitting. A Scatchard plotting for a system of this type is nonlinear. In the case of the competitive binding of two different ligands a t the same binding sites, the corresponding equation (5)is
where Ki and Kzare the hinding constants of the competitor, and [C] is its concentration.
The studv described below mav he carried out as an adSpectrofluorometric ~ e a s u r e m e r i of t the Degree vanced laboratory experiment to illustrate the possibilities of Occupation of snectrofluorometric techniaues in a problem a s importani a s the binding of ligands to mac~omolecules.~ 6 ~ e - If a protein is marked with a fluorescent probe, then the fully, it will help fill the need for undergraduate experihindine of another lieand c o m.~ e t i n for e the same binding ments related to fluorometric techniques (2,3I. sites can be studied b;following the variation in the intensitv of the fluorescence due to displacement. Afluorescent probe is a substance whose fluorescence is significantly Binding of Ligands to Macromolecules modified on reversibly hinding to the macromolecule. The The degree of occupation ( r ) of a macromolecule by a fluorescent probe used in this study is l-aniline-8-naftaligand is known to be related to the concentration of free lene sulphonate (ANSI. ligand, [Ll, when the molecule has n equal and indeThe intensity of fluorescence I f of a substance in solupendent binding sites for that ligand. tion under specific conditions is expressed as follows.
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where r is the number of moles of bonded ligand per mole of macromolecule, a n d K i s the hinding constant. This equation can he linearized in different forms. Scatchard's provides the best fit to the experimental data (4).
where Q is the quantum yield of fluorescence; E is the molar extinction coefficient; c is the concentration; andKfis a proportionality constant, which includes geometric and sensitivity factors proper to the apparatus and the conditions of the measurement. This equation is only valid for a very dilute solution; good linearity generally is obtained between If a n d c for solutions with a n absorbance not above 0.05.
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Volume 71 Number 12 December 1994
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the modern student laboratory Quantifying the Degree of Binding
In the case of a ligand with an intensity of fluorescence that varies significantly on binding to a protein, the degree of binding can be quantified as follows. Three solutions are prepared in the corresponding cuvettes. One has no protein (simply buffer). Another has a great excess of protein, so we may assume that almost all the robe to be added will be bonded. The third has a lower conLentration of protein, so the equilibrium of the binding can be determined. Successive amounts of probe are added to all three, and the intensity of fluorescence is measured under identical conditions. In the case of the third cuvette, the intensity of fluorescence of the solution IT is equal to the sum of that from the free probe and that from the bonded probe. According to eq 5, we have IT = K&WF
+K A E B C B
(6)
where F and B correspond to the free and bonded ligand. Because the total concentration of the probe cT must be C~=CF+CB
(71
we have IT = K~F%(cT- CB) + KFBQBCB
(81
The measurement of the intensity of fluorescence in the absence of protein I I (cuvette 1)gives the expression
Figure 1. Uncorrected spectra of fluorescence emission (a) ANSHSAcomDlex (HSA.0.05 OIL: ANS. 3.3 x lo4MI. 1bI ANS (3.3x lod M,, (cr A N S - ~ ~ S A C O IHSA, ~ ~ 0.05 ~ ~ g L: A N S 3.3 lo4 M) ,n tne presence of cane ne 1.0 10 M. A SolJtlons In 0.01 M phosphate b-Her w In excifaflon at i.= 375 nm ano sl ts of 5 nm in excllauon ano 10 nm in emission. The ANS emission spectrum (b)was recorded with sensitivity 8 times higher than the other two. A
When all the probe is bonded to protein (great excess of protein in cuvette 21, the intensity of the fluorescence is
Taking as a basis eqs 8-10 and taking into account the fact that C$CT is the fraction of the probe bonded ( X ) , we get the following expression.
Once the value ofX is known, the concentration of free probe [LI can be determined from the total concentration [LIT,according to the equation
Moreover, the degree of occupation r will be given by the expression
where [PITis the total concentration of protein. With Competition If another substance-one that competes with the probe for the same binding sites-is added to the solution containing the probe, with the protein in low concentration, a displacement occurs. This gives rise to a variation in the A298
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'
A
intensitv of the emission of fluorescenceIS. In this case the fraction of probe bonded in the presence of the competitor can be calculated in a way totally analogous to that presented
Hence there are sufficient data to determine the parameters of the bindine. In the first d a c e those corresponding to the probe can be determinid by experiments canied out in the absence of a competitor. Once these values are obtained, the binding constants of the competitor can be determined with the data obtained in the oresence of the latter, by means of fitting the experimentai data to eq 4. In this study a procedure is proposed whereby the parameters of the binding of caffeine to human serum albumin (HSA) are determined by using fluorescent ANS as a probe. Experimental Methodology To begin it is necessary to study the spectroscopiccharacteristics of the substances involved in order to establish the working conditions. Figure 1shows
.
the unadjusted spectrum of fluorescence emission of the AN%HSAcomplex (curve a) t h e emission spectrum ofANS (3.3 lo4M) (curve b)
Fiaure 3. Dearee 1L1. (a1 ~" of occuoation rvs. free wobe concentration . .. in aosence of cane ne anb (b) n the presence of caneme. Cfrclesare 0~
Figure 2. Degree of occupation r vs. free probe concentration, [Ll. Circles are experimental points and lines are the best-fitcurves (eq 3),assuming(a)n1=1,m=l,(b)ni=l,m=2.(c)nt=i,m=3,(d)
n,=i,m=4.
.
the spectrum of ANS-HSA complex in the presence of caffeine (1.0 lo3 M)(curve c)
All are in 0.01 M phosphate buffer. The h of excitation is 375 nm, and in all cases the slits are 5 nm in excitation and 10 nm in emission. The emission soectrum of the ANS was obtained with a sensitivity 8 times'higher than the other two. Examination of the spectra gives evidence to the great variation in the characteristics of the emission spectrum of the ANS on bindine to the orotein. There is a displacement in the position of the maximum and, with g-reker relevance to-this study, a great variation in the quantum yield of emission, which enables its degree of binding to be quantified. Also, in the emission spectrum of the ANS-HSA complex in the presence of caffeine no modification was observed in the shape of the spectrum or in the position of the maximum, but there was a significant decrease in the intensity of emission. This decrease could be due to various causes (6) discussed below.
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An "inner filter" effect caused hv the caffeine is olausible. hut not present, because at the excitation wavelength (375 nm) there is no absorption by the drug The caffeine was also susoected of eausine a displacement in t t w wavelenglh a f t h r maximn nfthe tluurescerm spectrum ofthe prohc IISAuumplex. Ilnwewr, examination ofthr corresponding spectra (Fig I , demonrlrates [hat this IS not iu. T h e decrease m the intensity of fluorercenw can h r inter. prercd hy praposinga comprutian between care-.nc and ANS tbr ~ h same r hmdmg sites in the protem because thr prnhe has almost no fluorescence when free in solution
The results obtained by the procedure described below confirmthis interpretation. Procedure Solutions of lo3 M ANS and 2 lo3 M caffeine were preoared. a s well a s two solutions of HSA. one of high concentration (2 mg/mL) and the other of low concentration (0.1 mgImL), all in 0.01 M phosphate buffer, pH 7.4.
~
~
experimental po nts an0
lnes are me oest f 1 CLNes
Three cuvettes of equal size were prepared to measure the fluorescence. The first contained 1.5 mL of the high concentration HSA solution and 1.5 mL of buffer. Previous tests proved that this concentration is adequate for the ANS to be considered a s almost completelv bonded in the work concentrations range. (There is-a linear relationship between intensity of fluorescence and probe concentration., In the secon-d cuvctte 1.5 mL of bulter were added 111 1.5 ml. of the low concentration HSA solution. The third cuvette contained 1.5 mL of the low concentration HSAsolution and 1.5 mLofthecaffeine solutwn, so the concentration of this substance in the cuvette was 1.0 x lo3 M. Given that the intensitv of the fluorescence of the ANS in an aqueous medium is negligible a s opposed to that corresponding to ANS bonded to HSA, the use of a fourth cuvette with buffer is unnecessary. The measurements were taken with a n RF-540 Shimadzu spectrofluorometer thermostatted a t 37 "C. Successive amounts of 6 uL of the ANS "matrix" solution were added to each of the cuvettes with a micropipet (Gilson). I n each case the intensity of fluorescence was measured, with excitation a t h = 375 nm and measurement of the emission a t h = 470 nm. After every addition each cuvette was stirred. (The stability of the intensity of the fluorescence had been confirmed ~reviouslva t 10 min, so the binding equilibrium was assumed to be established.) Then the measurement was recorded. In each experiment 11-13 additions of ANS were made to each cuvette. eivine orobe concentrations of 6 8 x lod M. Given the sh;all &ume added it was unnecessary to make any adjustment of the concentrations due to the dilution effect. Data Treatment With the data obtained the values of the fractions of probe bonded can he calculated, both in the absence and in the presence of caffeine using eqs 11 and 12, taking into account the fact that, given the characteristics of the emission of ANS, the ZIvalues are negligible. Once the values of r and [LI have been calculated using eqs 12 and 13, the values of the numbers of binding sites and the binding constants can be obtained for both ANS and caffeine. A Scatchard plotting (nonlinear) of the reVolume 71
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the modern student laboratory sults shows that the binding sites of ANS in HSA are not equal. On the contrary, a good fit to eqs 3 and 4 is obtained. . For the case when only Ahs' is present (absence of caffeine), Figure 2 shows the experimental results obtained with the curves corresponding to the fitting, by a nonlinear regression calculation program, to eq 3. The fit was carried out with the MULTI program (71,assuming whole values for the binding sites. The best result was found to be obtained for nl = 1 and n2 = 3. With these values binding constants were obtained for the ANS of Kl = (4.2 f 0.3) x lo6 and Kz = (5.2 f 0.4) x lo5. For comparison, curves with other nl and n2 parameters are also shown. Once these ~ a r a m e t e r sare determined. the fitting of the experimental data corresponding to the keasurem&ts taken in the presence of caffeine makes it possible to obtain the values of the binding constants of that substance: K; = 300 f 30 and K: = 4800 _+ 200. (All values of binding constants represent averages from three trials.) The best fitting is shown in Figure 3. Conclusions Through the use of a relatively s i m d e ~rocedurethis l the stuheni experience in laboratory cxpenmcnt w ~ l gwe an mumnnnt sub~ectin b~omedlcslsciences: the binding of small~moleculesto proteins. An interesting aspect of spec-
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Journal of Chemical Education
trofluommetry-the dependence of fluorescence emission properties upon the fluorophore environment-is illustrated. Students also learn to obtain quantitative information relative to the binding process from the observed variations. Chemicals used do not present any special safety problem, so only ordinary safety procedures need be followed. The laboratory work can be carried out in three sessions of approximately 3 h each. The first one can be dedicated to the ~ r e ~ a r a t i oofnsolutions and the studv of their soectroscopic characteristics, in order to select the working conditions. The second one may he used for carrying out the necessary meassurements for the binding study that would be convenient to make them threefold. The third session would be dedicated to the mathematical treatment of experimental data and the corresponding discussion of results. The time required for this last session could be less than for the other two. Literature Cited1. Weber. G.; Young,L.9. J B i d Chem 1964,219,1415. 2. Bower. N. W J C h m . Edue. 1982.59.975, 3. Hu. J.; Snkbei1.E. G.:White,H.B. J. Chcm.Educ.1990,67,803. 4 . Dunford, H. 9. J Cham. Edue 1984,61,129. 5. Seatchard. G.Ann. N Y Acod. Sci. 1949,51,660. 6. Lakowicz, J. R. P?'inciple~olFluomscence Spectmscom; Plenum Ress: New York,
.-"".
*"*,
7. Yamaoka, K:Tanigawara,X Nakagawa, T.; Uno, T. J
Phorm. Dyn.1981,4,879.
