Proc. Indian Acad. Sci. (Chem. Sci.), Vol. 99, No. 4, October 1987, pp. 273-279.
9 Printed in India.
Binding o f n a p r o x e n to bovine s e r u m a l b u m i n and tryptophan- modified bovine serum a l b u m i n
MEENAKSHI M A R U T H A M U T H U * and S KISHORE
Department of Physical Chemistry, University of Madras, Guindy Campus, Madras 600 025, India
MS received 17 November 1986; revised 21 August 1987
Abstract. Two classes of binding sites, a single high-affinity site with an association constant of 4.8 x 106 M ~ and two low-affinity sites with association constant of about 0.05 • 106 M - t have been observed in the interaction of Naproxen with bovine serum albumin (BSA). Chemical modification of two tryptophan residues in BSA with 2-hydroxy-5-nitrobenzyl bromide has led to a reduction in the association constant of the high-affinity site by 89% and its number of binding sites by 66% suggesting the involvement of tryptophan residues in the high-affinity site. In contrast, the two low-affinity sites were not affected by the modification. Binding of Naproxen to the low-affinity sites of BSA induces microdisorganisation of the albumin structure leading to conformational changes as evident from fluorescence measurements with 1-anilino-8- naphthalenesulphonic acid as the probe.
Keywords. Binding; Naproxen; bovine serum albumin; dialysis; chemical modification;
fluorescence; eonformational changes.
I. Introduction
Albumin is the predominant protein which binds drugs in blood and interstitial fluid (Peters 1976). The binding of a drug to proteins in the blood will strongly influence its distribution, elimination and pharmacological effect (Jusko and Gretch 1976; Martin 1965). The free concentration of a drug in plasma, on which the pharmacological activity of the drug depends, is determined by the association constant K, the number of binding sites n, and the total concentrations of the drug and the binding protein. Since the albumin molecule represents a homogeneous, well-defined and extensively studied protein with receptor-like properties (Muller et al 1978; Muller and Wollert 1979), bovine serum albumin (BSA) is an excellent model to study the molecular aspects of ligand-protein interaction.
The non-steroidal anti-inflammatory drug Naproxen is extensively bound to serum proteins with binding degrees above 99% (Mortensen et al 1979). A previous study of Naproxen-BSA interaction revealed that the binding of Naproxen to BSA is not affected by temperature and pH variations (Kaneo et al 1981). Besides this, detailed studies have not been made to investigate the specificity, the nature of binding sites or the amino acids involved. So it is of particular interest to
*To whom correspondence should bc addressed. 273
characterize the binding of Naproxen to BSA using native BSA and chemically modified BSA (with 2-hydroxy-5-nitrobenzyl bromide) and to foresee the possible role of tryptophan residues in Naproxen binding. The fluorescent probe 1-anilino- 8-naphthalene sulphonic acid (ANS) has been used in this study to get information on possible conformational changes of BSA induced by Naproxen.
2. Experimental
Bovine serum albumin (BSA) (essentially fatty acid-free) and 1-anilino-8- naphthalenesulphonic acid (ANS) were obtained from Sigma Chemical Co., USA.
Naproxen was a gift from Cipla Ltd. as pure sample. 2-hydroxy-5-nitrobenzyi bromide (I-Br) was obtained from Fluka. Dialysis bags (18/32) were obtained from Wilson Laboratories, USA. All other chemicals were of analytical grade.
Binding of Naproxen to BSA was investigated by equilibrium dialysis and fluorescence techniques. A!l experiments were carried out at pH 7-4 in 0.05 M NazHPO4-KH2PO4 buffer. BSA concentration was 9-17/xM. The initial' Nap- roxen concentration, [Naproxen]0 ranged from 3-0/zM to 24.0/xM.
The fluorescence measurements of the solution containing ANS-BSA with or without Naproxen were determined using Aminco-Bowman Spectrophotofluoro- metor with excitation and emission wavelengths of 375 nm and 475 nm respective- ly. In these solutions, the concentration of BSA (10.0/zM) was taken in large molar excess over ANS (0-8 ~M) so that ANS could bind only to its high-affinity site and its binding to its low-affinity sites is negligible. The molar ratio of Naproxen to BSA was varied from 0-2:1 to 8: 1. The intrinsic fluorescence of tryptophan residues in BSA, urea-BSA and modified BSA was observed with excitation at 280 nm and emission at 350 nm.
Modification o f BSA with l-Br: The two tryptophan residues in BSA were chemically modified at room temperature following the method of Karkhanis and co-workers (Karkhanis et al1975; Koshland et al 1964) using 10 M urea and 2000-fold molar excess of l-Br. A control experiment was carried out in the same way without l-Br and this BSA is called "urea-BSA".
3. Results and discussion
Bovine serum albumin (BSA) contains two tryptophan residues and is modified selectively with 2-hydroxy-5-nitrobenzyl bromide (l-Br), as described in w 2, and is a saturable process. The degree of modification can be determined from the ultraviolet absorption quotient as described (Karkhanis et al 1975), from which the number of residues of 1 incorporated in BSA is found to be 2.5, suggesting that the modification of two tryptophan residues is selective and quantitative. Figure 1 shows that the absorbance of BSA and urea-BSA are similar, whereas that of tryptophan-modified BSA (I-BSA) is increased between 250 nm and 350 nm corresponding to the amount of reagent incorporated and a maximum at 410 nm due to the presence of free reagent. Figure 2 shows the intrinsic tryptophan fluorescence of BSA, urea-BSA and I-BSA. When compared to native BSA, the
Binding of naproxen 275
0.88 0.75
0-50
,~ 0 . 2 5
250 280 410 500
A ( n m )
F i g u r e I. . U V - a b s o r p t i o n s p c c t r a o f B S A ( ( - o - o - o - ) . [ B S A ] = 16.11/.tM.
t
5 O
g
'!
u.
fo
o ;
3t30 350 4 0 0 4 5 0 X ( r i m )
). u r e a - B S A ( . . . ) a n d 1-BSA
F i g u r e 2. F l u o r e s c e n c e e m i s s i o n s p e c t r a o f B S A ( ), u r e a - B S A ( - o - o - o - ) a n d I - B S A ( - • - ~ - A - ) . T h e s p e c t r a w e r e r e c o r d e d u s i n g a n e x c i t a t i o n w a v e l e n g t h o f 280 n m . [ B S A ] = 9.17 p.M.
intrinsic fluorescence of urea-BSA is decreased by 18%, whereas it is reduced by 94% for I-BSA. The observed 18% decrease in urea-BSA is due to unfolding in acid solution, while the 94% decrease in I-BSA results from the modification of two tryptophan residues.
When the binding data of the BSA-Naproxen system, in the initial concentration range of Naproxen 3-0 p.M to 24-0 k~M, are plotted according to Seatchard (1949) in terms of r/C versus r (r = number of moles of drug bound per mole of BSA and C = [Naproxen]frcc) a curve was obtained (figure 3) indicating more than one class of binding sites. Graphical resolution according to the method of Pennock (1973) yields two classes of binding sites, one of high-affinity and the other of low-affinity with binding constants and number of binding sites K1, nl and K2, n2, i'espectively.
The binding parameters determined are reported in table 1.
1G
~4
12
10
"7
x e
o
6
ScatchQrd p l o t
0.5 I-0 1.S 2.0 2.S 3,0
Figure 3, Scatchard plot for the binding of Naproxen to B S A ( - o - o - c - ) , u r e a - B S A ( - [] - D - u - ) and I-BSA ( - A - A - • - ) r = mol d r u g b o u n d / m o l BSA.
C = e q u i h b r i u m concentration of the drug. [BSA] = 9.17 ~.M. [ N a p r o x e n ] , = 3-0 to 24-0/_tM.
Table I. Binding p a r a m e t e r s , T h e values a r e calcu- lated f r o m the Scatchard plot (r/C versus r) applying P e n n o c k ' s m e t h o d (1973) p H = 7.4; T e m p , = 25~
[BSA] = 9,17 txM [Naproxen]o = 3.0/~M to 24.0/~M.
Kl • 10 -6 K2 • 10 -6
(M -1) nl (M -1) nz
B S A 4.800 1.00 0-050 2-00
U r e a - B S A 3.420 0.76 0-051 2.24
I - B S A 0-529 0.34 0.037 1.60
From table 1 it can be seen that Naproxen has one high-affinity site with K~ = 4.8 x 10 6 M - 1 and two low-affinity sites with K2 = 0.05 x 106 M -1 in native BSA. Naproxen is one of the drugs strongly bound by serum proteins (Mortensen et al 1979). Strongly bound drugs have been shown to posses "receptor-like"
specificity (McMenamy and Oncley 1958; Muller and Wollert 1973) and it is reported that this specific nature is mediated by only a small number of binding sites (Sudlow et al 1975, 1976). Hence, the high K~ value observed represents the strong single binding site in BSA. When K1 and//2 values are compared, it is found that the Kt value is 96 times greater than the//2 value. This difference in K values is manifested not only due to the vast difference in the affinity of the two classes of
Binding of naproxen 277 sites, but also due to the dissimilar specificity and structural requirements. From the association constant values it can be deduced that the high-affinity site is highly specific and its structural characteristics are different from the two low-affinity sites.
When Naproxen binds to urea-BSA, KI and n l values decrease to 3-42 x 10 6 M - E and 0.76, respectively (table 1). The reduction seems to be of low significance as there would have been a larger decrease if the specificity of the high-affinity sites were lost. The decrease is attributed to acidic unfolding in urea solution at pH 4.4 and an incomplete refolding when the pH is raised to 7.4. The values of K2 and n2 are found to be unaltered.
When Naproxen binds to I-BSA, in which the two tryptophan residues are chemically modified, K1 and nl values are reduced to 0.529 x 106 M -I and 0.34, respectively. The observed decrease of 89% and 66% in K1 and nl values, respectively, occurs due to the loss of the affinity of the site. Naproxen is an arylpropionic acid drug.
CH 3 / ~ C H - I COOH
CH30~ v v
Naproxen
The presence of a hydrophobic area in BSA is essential in order to have a strong hydrophobic interaction with Naproxen. Further, the inevitable presence of the one tryptophan residue in this hydrophobic area (since tryptophan is the most nonpolar of all amino acids) is necessary to provide the needed specificity, hydrophobicity and structural requirements. The specific and quantitative modi- fication of these residues results in the loss of specificity and structural characteristics leading to the observed reduction in K~ and n~ values. Hence the presence of tryptophan fulfils the structural features required for the binding of Naproxen to its high-affinity site.
The K2 and n2 values for the binding in I-BSA are reduced by 26% and 20%, respectively, which precludes the possibility of the involvement of the tryptophan residues in the low-affinity sites. The difference in the characteristics and the specificity of the two classes of sites is confirmed as the structural requirements of the two low-affinity sites do not require the presence of the tryptophan residues to be part of the sites.
Conformational changes induced by Naproxen: Changes in the conformation of albumin as a result of binding of small molecules has been observed previously (Blanchar et al 1975; Krieglstein 1969; Scholtan 1964; U.chida and Hanano 1974), and these changes were detected by monitoring the fluorescence of bound probes.
Fluorescent probe techniques monitor only small areas of protein molecule and hence conformational changes observed may be relatively localised. In order to monitor the conformational changes, the limiting condition at which the probe is completely bound to BSA is used. The probe used in this study is 1-anilino-8- naphthalene sulphonic acid, which binds preferably to the hydrophobic site of the BSA. ANS-BSA emits at 475 nm when excited at 375 nm. When the ratio
40O
35O
300
o 2 5 0
200 o
~'50 c t i I
O -
i i
, i I I I I I I 0
10 20 30 40 50 60 70 B
FNoproxen-], ~ M
Figure 4. Effect of increasing concentration of Naproxen on the fluorescence of ANS-BSA complex. [ANSI = 0-8/zM; [BSA] = 10-0/zM. Excttation and emission wavelengths are 375 nm and 475 nm respectively,
[ANS]/[BSA] is 0-08, where the limiting condition is obeyed, it represents a condition, sensitive to the changes in protein structure around the binding sites of the probe as observed elsewhere (Birkett e~ al 1977).
Increasing amounts of Naproxen were added to ANS-BSA mixture at the limitin; condition, such that drug to BSA ratio varied from 0.2:1 to 8:1. The fluorescent emission was unaltered by addition upto the drug: BSA ratio of 0.8:1 (figure 4), at which value Naproxen would bind to its high-affinity site only. The microenvironment which is sensitive under this limiting condition is unaltered leading to this negligible change. Further addition of Naproxen upto a drug: BSA ratio of 3:1, when the drug also binds to its low-affinity sites causes increase in fluorescence by 200% (figure 4), above which further addition causes negligible increase. The occupation of the two low-affinity sites by Naproxen causes conformational changes in protein (BSA) structure around ANS-BSA binding site resulting in the microdisorganisation of the ANS-BSA microenvironment leading to predominant increase in fluorescence.
Thus the binding of Naproxen to its high-affinity site does not cause conformational changes, whereas binding to its low-affinity sites leads to disorganisation of the microenvironment resulting in Iocalised conformational changes. The above mentioned difference between the two classes of sites confirms the tryptophan modification studies, where distinction arises out of dissimilar structural specificities of the sites, the high-affinity site requiring tryptophan residues to be part of its site whereas the two low-affinity sites do not require tryptophan residues for the binding.
Acknowledgement
This work was supported by CSIR New Delhi. The authors wish to thank Cipla Ltd., India for providing a pure sample of Naproxen.
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