Fluorometric Determination of Drug-Protein Association Constants: The Binding of 8-Anilino- 1-naphthalenesulfonate by Bovine Serum Albumin Datta V. Naik, W. Larry Paul, Rose Marie Threatte, and Stephen G. Schulman' College of Pharmacy, University of Florida, Gainesville, Fla. 326 10
The binding of 8-anilino-1-naphthalenesulfonate to bovine serum albumin was reinvestigated. A fluorescence method was used to obtain the binding data which were treated appropriately to obtain the number of binding sites and the equilibrium constants. The results confirm that the number of binding sites for 8-anilino-1-napthalenesulfonate on bovine serum albumin is 3. The individual binding constants were calculated by using the Bjerrum technique. The average values of the three constants are K , = 2.9 X lo6, K 2 = 6.1 X lo5, and K3 = 5.6 X lo5.
8-Anilino-1-naphthalenesulfonate(ANS) has been extensively used as a fluorescent probe for such studies as the quantitative analysis of serum proteins ( I ) , t h e study of the polarity of binding sites on proteins ( 2 ) ,and the determination of the protein-binding parameters of other drugs by competitive binding (3, 4 ) . Rees and coworkers ( I ) reported the use of ANS for the assay of serum proteins. However, it was Daniel and Weber ( 5 ) who studied t h e interaction between ANS and bovine serum albumin (BSA) in detail, They reported that the interaction was reversible with a n average binding constant of about lo6, and that five molecules of ANS were bound per molecule of BSA. Recently Ma, J u n , and Luzzi (6) have published new data on ANS-BSA binding. Their value for the average binding constant agrees well with t h a t reported by Weber. However, they found t h a t the maximum number of binding sites, N , on BSA, for ANS was three rather than five. Because of these conflicting results, it was decided to reinvestigate the ANS-BSA binding interaction.
EXPERIMENTAL Sodium 8-anilino-1-naphthalenesulfonatewas purchased from Eastman Organic Chemicals, Rochester, N.Y. The sodium salt was purified by passing an aqueous solution of the salt through an Amberlite IR-120 (Mallinckrodt) column, and then further purified by crystallization from water. Crystalline bovine serum albumin of A grade (Pentex brand product of Miles Laboratory) was supplied by Calbiochem, San Diego, Calif. Commercially prepared BSA usually contains fatty acids as impurities and can be purified by acid charcoal treatment ( 7 ) . However, Chen ( 7 ) has shown that Pentex brand BSA contains only about 1 mole of fatty acid per mole of BSA. Moreover, Santos and Spector (8) have reported that up to 2 moles of long chain fatty acid per mole of BSA do not affect the ANS-BSA binding. Consequently, the pretreatment of BSA with acid charcoal was deemed unnecessary. Because of the hygroscopic nature of BSA, it has been common practice not to accurately weigh samples routinely for dissolution in water, but rather to photometrically determine the absorbance at 280 nm of aqueous solutions containing roughly weighed BSA samples. The concentration in g/l. is obtained from Beer's law, assuming a value of E ;Trnof 6.60 ( 4 ) or in moles/l. by assuming as well the average molecular weight of 69,000 (9, IO) so that c = 4.55 X IO4 at 280 nm. In our experience, during the course of an average accurate weighing (in the extremely humid Florida climate), an error of no more than 1%was Author to whom correspondence should he addressed.
incurred, by water absorption by BSA. Moreover, the results obtained in these experiments were identical regardless of whether BSA concentration was determined photometrically or directly by weighing. It appears that photometric standardization of BSA solutions is superfluous. All BSA and ANS solutions were prepared two hours prior to fluorometric titration, from distilled, deionized water buffered to pH 7.4 with a total phosphate ([H2P04-] + [HP042-]) concentration of 0.1M. The fluorometric titrations were carried out as follows: 2.0 ml of the protein solution of appropriate concentration in l a 1-cm silica cell were titrated by successive additions of l - ~ volumes of 1 X 10-3M solution of ANS, delivered from a Unimetrics (air and liquid tight) microsyringe. The temperature during the titrations was maintained at 25 "C. The fluorescent intensity measurements were made on a Perkin-Elmer MPF-2A fluorescence spectrophotometer with excitation at 360 nm and fluorescence monitored at 464 nm.
DISCUSSION The fluorometric titrations of BSA with ANS are shown in Figure 1. T h e fluorescence intensities for the two titrations with high protein concentrations (curve a ' ) 2.5 X 10-5 and 5 X 10-5M, were identical, indicating that the ANS added was fully bound a t both the protein concentrations. T h e previous investigators ( 6 ) have shown that, at least approximately, the first three binding constants are very close. Therefore, it is reasonable t o expect that, even in the presence of excess protein, the bound ligand would exist as BSA(ANS)l, BSA(ANS)z, and BSA(ANS)3. T h e doubling of total protein concentration must result in a substantial redistribution of the ligand among the various complexes. Since the fluorometric titrations coincide for both high protein concentrations, it may be concluded that, within experimental error, the quantum yield of fluorescence, and therefore the fluorescence intensity of the bound ligand, is constant and independent of the stoichiometry of the corn-' plex in which the ligand resides. This conclusion suggests that the binding sites for ANS on BSA are widely separated and very weakly interacting and is supported by the existence of an isosbestic point, a t 360 nm, in the absorption spectrum of the ANS-BSA system when a constant amount of ANS is titrated with BSA. For substrates like ANS, which fluoresce much more intensely when bound than as the free ligand, the fraction of substrate bound, a, is usually determined by using the equation (11 ):
a=-
F, - F F, - F
where F and F are the fluorescence intensities of a given concentration of substrate in solution of low protein concentration and in solution without any protein, respectively. ( F essentially represents the background fluorescence.) F b is the fluorescence intensity of the same concentration of the fully bound probe. T h e latter is taken to be the fluorescence intensity of the probe in the presence of excess protein. Such a treatment will yield good values of cy provided the fluorescence intensity of the bound substrate is a
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A N S C O N C E N T R A T I O N , x106Y
Figure 1. Plots of relative fluorescence intensities as a function of total ANS concentration for the BSA-ANS titrations with constant amount of BSA. Curve a', [BSA] and 5 X lo+; curve b, rBSA1 . . = 2.5 X . . = 2.5 X 10-6M; and curve c, [BSA]= 1 X lO-'M. ing the fluorescentintensities in curve a' for the absorbance effect (see text)
linear function of its concentration. The linearity of F b with substrate concentration can only be taken for granted a t very low absorbance ( 0.02, the deviation from linearity will be greater than 2%. A t absorbances of 0.02 < A < 0.15, the second term in Equation 2 results in 115 X A % deviation from linearity a t any point in the fluorometric titration. At higher absorbances, the third term in Equation 2 may have to be considered. A t the excitation wavelength, 360 nm, the molar absorptivity for the ANS-BSA system was determined to be 6 x lo3 liter mole-' (ANS) cm-l. Hence a t ANS concentration of greater than 3.3 X 10-6M (A = 0.02), the deviation from linearity will be greater than 2%. It is therefore inappropriate to use Equation 1 to calculate the concentration of bound ANS. For the titrations with high protein concentration, the observed fluorescence intensity a t every point during the titration was corrected for the second term in Equation 2 by applying the correction factor 1.15A. Curve a', in Figure 1, was plotted using the observed values of Fb - F. After applying the correction to the observed fluorescence intensities, a straight line plot (line a in Figure 1)was obtained, verifying t h a t the deviation from linearity of curve a' was indeed due to the absorbance effect. When titrations are carried out a t low protein concentration so that the substrate would be only partially bound, the absorbance, A , in Equation 2 is the difference between observed absorbance and the absorbance due to the unbound ANS concentration, because it is only the bound ANS t h a t is responsible for fluorescence. Thus the points 268
The straight line a is obtained after correct-
in the fluorometric titration curves b and c (Figure 1) cannot simply be corrected for nonlinearity of fluorescence intensity with concentration of bound ligand (absorbance) by application of Equation 2 (unless the bound substrate is the only absorbing species). Rather, it is convenient to use curve a as a calibration curve. T h e fluorescence intensity a t any point on curve b or c can be regarded as arising from the bound ligand concentration giving the same observed intensity on curve a'. For purposes of equilibrium calculations, the fluorescence intensity read from b or c can be made directly proportional to bound ANS concentration by comparing the fluorescence intensity in question on curve a' to the corresponding point (same concentration of ANS) on curve a. T o calculate the maximum number of binding sites, N, the method of Edsall e t al. ( 1 2 ) , as employed by Ma and coworkers (6), was first used. Edsall's treatment involves the use of a modified Scatchard equation ( 1 3 ) ,
where Q is a function of the equilibrium constant, fi equals the number of moles of substrate bound per mole of protein, N equals the maximum number of binding sites, and [D]equals the equilibrium concentration of free substrate. Plots of log Q us. iz are constructed with trial values of N . T h e value of N t,hat gives a straight line plot is taken to be the correct number of binding sites. If the N binding sites are identical and non-interacting (Le,, all successive binding constants are identical), the function Q will be a constant which is independent of fi, and be equal to the equilibrium constant. In this case plot of log Q us. f i will be a straight line with slope equal to zero, However, if the binding sites are interacting, the log Q will be a linear function of fi with a non-zero slope. Figure 2 shows the Edsall's plot for the ANS-BSA {[BSA] = 2.5 X 10-6M1 with trial values of N = 2 , 3 , 4 , and 5. Reasonably straight lines are obtained for N = 3, 4,and 5 . T h e line with N = 3 gives a near-zero
ANALYTICAL CHEMISTRY, VOL. 4 7 , NO. 2, FEBRUARY 1975
%FL
INT
0
N=2
0
0 0
MOLE F R A C T I O N OF A N S ,
I
I 0
I
, 1
,
,
-
2
,
xi
T O T A L CONC 1 0 . 5 ~
Figure 3. Job's Plot of relative fluorescent intensities as a function of mole fraction of ANS. The total concentration of [BSA] [ANSI was kept constant at 1 V 5 M
+
, 3
n
Figure 2. Plots of log Q vs. rS for the ANS-BSA titration {[BSA] = 2.5 X 10-6M) with different trial values of N, the number of binding sites
P + D
+D
PD slope while the other two lines have negative slopes. Assuming the binding sites are non-interacting, N = 3 seems to be a good choice. But N could be 4 or 5 if the sites were interacting. Thus, the exact number of binding sites cannot be ascertained unambiguously from this treatment, at least in the present case. The plateaus in the titration curves with lower protein concentrations (Figure 1) indicate the saturation of BSA binding sites. Tangential extrapolation of these curves should give the number of maximum binding sites, N . The extrapolation of the curve, c, for the titration with [BSA] = 1 x 10-fiM cannot be done, with any certainty, because of its continuous curvature a t low ANS concentrations. However, the plateau of this curve corresponds to a fluorescence intensity equivalent to a bound ANS concentration of 2.8 X 10-fiMM,and hence an integral value of N can be approximated as 3. The extrapolation of the curve, b, for the titration with BSA = 2.5 X 10-fiM is feasible, and gives a value of N = 3.0. T o further establish the value of N , a Job's plot ( 1 4 ) was carried out for the ANS-BSA system by keeping the total concentration of ANS plus BSA a t 1 X lOP5M. This plot is shown in Figure 3. The inflection point gives a value of 0.76 which corresponds to N = 3.1. Therefore, it is reasonably certain t h a t the maximum number of binding sites, on the BSA molecule for ANS is 3. Protein binding equilibria have traditionally been evaluated by means of the Scatchard equation, (4) or its modified forms. One of the main assumptions made in the use of the Scatchard equation is that the N binding sites are identical and non-interacting, which in reality may not be true. I t occurred to us that the binding of a substrate to a protein is similar to the formation of a metal-ligand complex. The exact formation constants of metal-ligand complexes are generally calculated from the Bjerrum technique (15). This approach is applicable to any system in thermodynamic equilibrium and makes no presupposition concerning the equivalence of binding sites or relative magnitude of successive equilibrium constants. The three successive equilibria:
Ki
==
PD
K2
=== P D ,
K3 +D +
PD,
PD,
where P is protein and D is a substrate, are characterized by equilibrium constants K 1, Ka, K3, such that
(5)
Assuming that the p H is such that the predominant prototropic species in solution is the ligand itself (which is the case for ANS a t p H 7.4) the total substrate concentration, D T,is given by
DT = [ D ]
+
[PD]
+
2[PDz]
+
3[PD,]
(8)
and the total protein concentration, PT,is
PT = [PI
+ [ P D ] + [PD,] + [PD,]
(9)
Combining Equations 5, 6, and 7 with Equations 8 and 9 yields
D, = [D]
+
K i [ P I [ D I + 2KiKz[PI[D12 + 3KiK2K,[P][D13 (10) and similarly
P T = [PI
+ K i [ P I [ D I + K1Kz[PI[D12 + KtKzK3[PI[D13 (11)
The average number, R, of bound substrate molecules per protein molecule is
(12) Combination of Equations 10, 11,and 12 gives
ANALYTICAL CHEMISTRY, VOL. 47, NO. 2, FEBRUARY 1975
269
~
Table I. Equilibrium Constants for the Binding of 8-Anilino-1-naphthalenesulfonate to Bovine Serum Albumin at 25 "C -
Protein conccn t r a t i o n , mol'l.
K 3
K1
2. 5 x 10'6
2.8 i 0.6 x 106
1.0 x 10-fi
3.1
i 0.7
x 106
B2 = KIKz
1.7
0.5 x 1012 1.9 0.5 x 10'2
B 3 = K K,,H
(7-2 -
l)K,[D]
6.1 x 10'
5.9 x 10'
1.0 x 106
6. 1 x 10'
5.3 x 10'
1.0 x lofi
*
i
For any value of [ D ](determined from Figure I), a value of fi when substituted into Equation 14 generates a linear equation with three unknowns, which can be solved graphically to get an estimate of K 1, &, and p3. These values are then refined by fitting to Equation 14. A simpler treatment is possible; but only if the individual constants are widely separated (at least 2 orders of magnitude) such that only one equilibrium is experimentally detectable (to within 99%) a t any point in the titration. In this case, a plot of -log [ D ] us. fi would show (for the ANS-BSA system) three distinct inflection regions which would correspond to -log [ D ]= log K 1 when fi = 0.5 -log [ D ]= log K P when i2. = 1.5 -log [ D ] = logK3 when = 2.5 Figure 4 shows the Bjerrum plot of -log [D] us. fi for the ANS-BSA titration ([BSA] = 2.5 X 10-6M). Clearly, the absence of sharp inflection points indicates strongly overlapping successive equilibria, and hence the analytical fitting of the data to Equation 14 is required if reasonably accurate constants are to be obtained. Table I lists the constants calculated in such a manner for both of the titrations
270
* 0.5
x 10'8 1.0 + 0 . 5 x 1018
Rearrangement of Equation 13 gives
+
K3
1.0
n
n
KZ
f
Figure 4. Bjerrum plot o f -log [D]as a function of ii for the BSAANS titration with [BSA] = 2.5 X 10-6M
-
l " 3
(curves b and c in Figure 1) a t lower protein concentrations using the data corrected for the nonlinearity in fluorescence intensity. The three constants obtained for each of the two titrations are similar within experimental error, indicating that the binding constants are independent of total protein concentration. The value of the average equilibrium constant, K, of 1.0 X lo6 is comparable to the values reported earlier (5,6).Daniel and Weber ( 5 )in their study of the ANS-BSA system had observed that the Bjerrum plot for the binding of ANS to BSA a t pH 7.0 and a t 25'C reveals a slight inflection a t about fi = 1 and a smooth curve thereafter. However, they did not attempt a detailed analysis of the binding constants. The presence of a slight inflection around fi = 1 and its absence for A > 1 indicates appreciable difference between the magnitude of K1 and K Z and similarity in the values of Kz and the rest of the equilibrium constants. I t is interesting to note that our results also show K1 to be higher, by about 0.7 log unit, than Kz while K2 is equal to K:j within the experimental error. The significance of this result may be important, but it is too early to make any definite conclusions about the order of magnitude of the equilibrium constants. Further studies on protein binding of compounds similar to ANS are in progress in this laboratory. LITERATURE CITED (1)V. H. Rees, J. F. Fildes. and D. J. R. Laurence, J. Clin. Pathol., 7, 336 (1954). (2)D. C.Turner and L. Brand, Biochemistry, 7, 3381 (1968). (3)T. Kinoshita, F. Linuma, lkuo Moriguchi, and Akio Tsuji, Chem. Pharm. Bull.. 19, 861 (1971). (4)H. W. Jun, L. A. Luzzi, and P. L. Hsu, J. Pharm. Sci., 61, 1835 (1972). (5) E. Daniel and G. Weber, Biochemisfry, 5, 1893 (1966). (6)J. K. H. Ma, H. W. Jun, and L. A. Luzzi. J. Pharm. Sci., 62,2038 (1973). (7)R. F. Chen, Biol. Chem., 242, 173 (1967). (8)E.C.Santos and A . A. Spector, Biochemistry, 11, 2299 (1972). (9)N. K. Patel, P. C. Shen, and K. E. Taylor, J. Pharm. Sci., 57, 1370 (1968). ( I O ) J. K. H. Ma, H. W. Jun, and L. A. Luzzi. J, Pharm. Sci.. 62, 1261 (1973). (11)G.WeberandL. B. Young, J. Bio/. Chem., 239, 1415 (1964). (12)J. T. Edsall, C. Felsenfeld, D. S. Goodman, and F. R. N. Gurd, J. Amer. Chem., SOC.,76, 3054 (1954). (13)G.Scatchard, Ann. N.Y. Acad. Sci., 51, 660(1949). (14)P. Job, Ann. Chim. (Paris), 9 (lo),113 (1928). (15)J. Bjerrum, "Metal Ammine Formation in Aqueous Solutions," P. Haase and Son, Copenhagen, 1941.
RECEIVEDfor review June 17, 1974. Accepted October 14, 1974.
A N A L Y T I C A L CHEMISTRY, VOL. 47, NO. 2, FEBRUARY 1975
