Elucidation of binding mechanism and identification of binding site for an anti HIV drug, stavudine on human blood proteins B. Sandhya, Ashwini H. Hegde & J. Seetharamappa
Molecular Biology Reports An International Journal on Molecular and Cellular Biology ISSN 0301-4851 Mol Biol Rep DOI 10.1007/s11033-012-2460-8
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Author's personal copy Mol Biol Rep DOI 10.1007/s11033-012-2460-8
Elucidation of binding mechanism and identification of binding site for an anti HIV drug, stavudine on human blood proteins B. Sandhya • Ashwini H. Hegde • J. Seetharamappa
Received: 25 July 2012 / Accepted: 18 December 2012 Ó Springer Science+Business Media Dordrecht 2012
Abstract The binding of stavudine (STV) to two human blood proteins [human hemoglobin (HHb) and human serum albumin (HSA)] was studied in vitro under simulated physiological conditions by spectroscopic methods viz., fluorescence, UV absorption, resonance light scattering, synchronous fluorescence, circular dichroism (CD) and three-dimensional fluorescence. The binding parameters of STV–blood protein were determined from fluorescence quenching studies. Stern–Volmer plots indicated the presence of static quenching mechanism in the interaction of STV with blood proteins. The values of n close to unity indicated that one molecule of STV bound to one molecule of blood protein. The binding process was found to be spontaneous. Analysis of thermodynamic parameters revealed the presence of hydrogen bond and van der Waals forces between protein and STV. Displacement experiments indicated the binding of STV to Sudlow’s site I on HSA. Secondary structures of blood proteins have undergone changes upon interaction with STV as evident from the reduction of a-helices (from 46.11 % in free HHb to 38.34 % in STV-HHb, and from 66.44 % in free HSA to 52.26 % in STV–HSA). Further, the alterations in secondary structures of proteins in the presence of STV were confirmed by synchronous and 3D-fluorescence spectral data. The distance between the blood protein (donor) and acceptor (STV) was found to be 5.211 and 5.402 nm for STV–HHb and STV–HSA, respectively based on Fo¨ster’s non-radiative energy transfer theory. Effect of some metal ions was also investigated. The fraction of STV bound to HSA was found to be 87.8 %. B. Sandhya A. H. Hegde J. Seetharamappa (&) Department of Chemistry, Karnatak University, Dharwad 580003, Karnataka, India e-mail:
[email protected]
Keywords Stavudine Human hemoglobin Human serum albumin Binding parameters Secondary structure Introduction Human hemoglobin (HHb) and human serum albumin (HSA) are the two major blood protein components in human circulatory system and erythrocytes. These can reversibly bind several endogenous and exogenous agents such as drugs. Characterization of the interaction of drug molecules with such proteins therefore constitutes a key step in drug development. The essential part of HHb contains two identical a-chains of 141 amino acids each and two identical b-chains of 146 amino acids. Each a-chain is in contact with the b-chain [1]. HHb has three tryptophan units (a214Trp, b215Trp and b217Trp) in each a and b chain (Protein Data Bank, PDB 2h35) [2]. HHb is an important functional protein for reversible oxygen carrying and storage; the potential changes of conformation and function of HHb upon binding to drugs has been a core of study. HSA consists a single chain of 585 amino acids and folded into three homologous domains (I, II and III), each of which contains two subdomains (A and B), and is stabilized by 17 disulphide bridges. It has two primary binding sites, site I and site II, which are hydrophobic cavities located in subdomain IIA and subdomain IIIA, respectively. The sole tryptophan residue (Trp 214) of HSA is in subdomain IIA. Most compounds bind to these sites. Stavudine (Fig. 1), a structural analog of thymidine, was firstly synthesized by Jerome Horwitz [3, 4]. It is widely used as a nucleoside analog reverse transcriptase inhibitor (NARTI) active against HIV [5]. STV caused serious and possibly deadly damage to the liver and pancreas and a lifethreatening condition called lactic acidosis [6].
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Author's personal copy Mol Biol Rep
Procedures
O
CH3
Fluorescence quenching measurements
HN
N
O
O HO Fig. 1 The structure of stavudine (STV)
The interaction of STV with HHb has not been investigated so far. However, thermodynamics of ferric heme binding to HSA at 25 °C in phosphate buffer of pH 7.0 in the absence and presence of STV were reported [7]. Further, Bocedi et al. [8] have studied the intrinsic tryptophan fluorescence of HSA upon excitation at 280 nm. These reports deal with interactions of a group of anti-HIV drugs with protein. They have not carried out the interactions of STV with protein in detail. In view of this, we have focused only on STV and its interaction with two types of blood proteins. Different aspects of STV– protein interactions viz., quenching mechanism, binding force operating between the drug and protein, the distance of separation between the protein and STV (based on the theory of fluorescence resonance energy transfer), conformational changes etc. have been studied.
Materials and methods Materials HHb and HSA were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and used without further purification. STV was obtained as a gift sample from Vivin Laboratories Pvt Ltd, India. All chemicals used were of analytical reagent grade and Millipore water was used to prepare the solutions. A stock solution of STV (250 lM) was prepared in phosphate buffer of pH 7.4. Solutions of HHb and HSA were also prepared in phosphate buffer of pH 7.4.
The excitation wavelength of 280 nm for HHb and 296 nm for HSA was used. The emission spectra were recorded from 280 to 500 nm for HHb and from 300 to 500 nm for HSA at three temperatures (288, 298 and 308 K). The concentration of protein was fixed at 2.5 lM while that of the drug was varied from 5 to 45 lM. Displacement experiments for the binding site of drug in HSA Displacement experiments were performed using different site probes viz, phenylbutazone, ibuprofen and digitoxin for site I, II and III, respectively [9, 10]. The concentration of protein and site probe was fixed at 2.5 lM and that of the drug was maintained as before in fluorescence quenching experiments. Resonance light scattering (RLS) studies RLS were obtained by synchronous scanning with the wavelength range of 250–500 nm keeping the Dk (kem - kex) = 0 nm on the spectrofluorometer at room temperature. Cprotein = 2.5 lM and CSTV = 5, 10 and 15 lM. UV absorption measurements The UV absorption measurements of HHb/HSA in the presence or absence of STV were made in the range of 200–600 nm and 240–320 nm for HHb and HSA at 298 K. For this, the protein concentration was fixed at 2.5 lM while that of the drug was varied from 5 to 45 lM. CD measurements CD spectra of HHb/HSA in the presence or absence of STV were recorded in the range of 200–250 nm with three scans averaged for each CD spectrum. For this, the molar ratio of protein to drug concentration was maintained at 1:1 and 2:1 for STV–HHb and at 0.5:1, 1:1, 2:1 and 3:1 for STV–HSA.
Apparatus Synchronous fluorescence measurements UV absorption spectra were recorded on a Hitachi U-3310 spectrophotometer (Japan) with 1.0 cm quartz cell. All fluorescence measurements were performed on a F-2500 spectrofluorometer (Hitachi, Japan) equipped with a thermostatically controlled cell holder and a 1.0 cm quartz cell. CD spectra were recorded on a Jasco J-810 spectropolarimeter (Japan).
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The synchronous fluorescence characteristics of STV– HHb/HSA were noted down at different scanning intervals of Dk (Dk = kem - kex). When Dk = 15 nm, the spectrum characteristics of protein tyrosine residues were observed and when Dk = 60 nm, the spectrum characteristics of protein tryptophan residues were noticed.
Author's personal copy Mol Biol Rep
3-D fluorescence studies
(A)
50
a
Intensity
3-D fluorescence spectrum was recorded under the following conditions: excitation wavelength range of 200–350 nm and emission wavelength range of 200–550 nm and an increment of 10 nm with other parameters were just the same as that of fluorescence quenching spectra. Cprotein = 2.5 lM and CSTV = 35.0 lM.
75
j 25
Influence of common ions on binding of STV with protein
X 0 280
The fluorescence spectra of STV–HHb/HSA were recorded in the presence and absence of various ions upon excitation at 280/296 nm. The concentrations of protein and common ions (K?, Co2?, Cu2?, Ni2?, Mn2? and Zn2?) were fixed at 2.5 lM while that of STV was varied from 5 to 45 lM.
350
420
490
Wavelength (nm)
(B)
150
Intensity
100
Results and discussion
a
50
j
Fluorescence quenching studies Blood proteins have strong fluorescence due to the presence of tryptophan units. Figure 2 shows the fluorescence spectra of HHb and HSA in the presence of different concentrations of STV. HHb and HSA have emission peaks at 348 and 351 nm, respectively. STV caused concentration dependent quenching of the intrinsic fluorescence of blood protein, without changing the emission maximum peak of HHb but with a slight blue shift for STV–HSA (351–348 nm). These results indicated that there were interactions between STV and blood proteins. In order to decide the type of quenching mechanism, the fluorescence quenching data were analyzed using the Stern–Volmer equation [11] shown below: Fo =F ¼ 1 þ Ksv ½Q
ð1Þ
where Fo and F are the steady-state fluorescence intensities in the absence and presence of quencher, respectively; Ksv is the Stern–Volmer quenching constant and [Q] is the concentration of quencher (STV). The values of Ksv can be obtained from the slope of Stern–Volmer curves. The linear regression plot of Fo/F versus [Q] (figure not shown) at every experimental temperature indicated that only one kind of quenching mechanism was predominant. The values of Ksv at different temperatures are shown in Table 1. The values of Ksv decreased with increase in temperature indicating the presence of static quenching mechanism due to the formation of STV–protein complex. The fluorescence quenching data were further analyzed according to the modified Stern–Volmer equation shown below:
X 0 300
350
400
450
500
Wavelength (nm)
Fig. 2 Fluorescence quenching spectra of A HHb (2.5 lM) and B HSA (2.5 lM) in the presence of increasing amounts of STV. a–j indicate the emission spectra of protein in the presence of 0, 5, 10, 15, 20, 25, 30, 35, 40 and 45 lM STV. (x) represents 5 lM STV only
Fo =ðFo F Þ ¼ 1=fa Ka ½Q þ 1=fa
ð2Þ
where Ka is the modified Stern–Volmer association constant and fa is the fraction of the initial fluorescence that is accessible to quencher. The plot of [Fo/(Fo - F)] versus 1/[Q] (figure not shown) yielded 1/fa as the intercept and 1/fa Ka as the slope. The values of 1/fa were found to be 0.8335 and 0.7206 for STV–HHb and STV–HSA, respectively indicating that only 83.35 and 72.06 % of the initial fluorescence of respective protein was accessible to quencher. The Ka values were calculated to be 8.49 9 105, 7.73 9 105 and 7.59 9 105 L mol-1 for STV–HHb and 6.99 9 105, 6.56 9 105 and 5.62 9 105 L mol-1 for STV–HSA at 288, 298 and 308 K, respectively. The decreasing trend of Ka with increase in temperature was observed to be in accordance with the dependence of Ksv on temperature. This is in consistence with static quenching mechanism as proposed above [12]. Equilibrium dissociation constant The equilibrium dissociation constant for STV binding to HSA was determined in the absence and presence of STV
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Author's personal copy Mol Biol Rep Table 1 Binding characteristics and thermodynamic parameters for STV–HHb and STV–HSA systems KSV (L mol-1)
K, 104 (M-1)
n
R
288
9.97 9 103
2.51
1.09
0.9980
298
8.85 9 103
1.89
1.07
0.9973
308
3
7.98 9 10
1.60
1.06
0.9970
288
1.40 9 104
2.22
1.06
0.9877
298
1.21 9 104
1.92
0.97
0.9994
308
1.03 9 104
1.36
0.93
0.9996
T (K)
DH° (k J mol-1)
DS° (J mol-1 K-1)
-64.41
-138.41
DG° (k J mol-1)
HHb -24.55 -23.16 -21.79
HSA
at different concentrations. The fluorescence quenching data were analyzed using the following equations; a ¼ 1=ð1 F=F0 Þ
ð3Þ
and a ¼ Kd =½Q þ 1
ð4Þ
where Kd is the equilibrium dissociation constant for the reversible binding of STV to HSA. The value of Kd (8.49 9 10-5 M) was obtained from the slope of the straight line plot of a versus 1/[Q] (figure not shown). The value of Kd was observed to be in excellent agreement with that of the reported (8.7 9 10-5 M); [8]. Accounting for the physiological concentration of HSA (7.0 9 10-4 M) [13] and the average anti-HIV drugs concentration in plasma (1.0 9 10-4 M) [14–17], the value of the molar fraction of the drug bound-HSA (W), the molar fraction of the drug free-HSA (X), the molar fraction of the HSA bound-drug (Y), and of the molar fraction of the HSA free-drug (Z) have been calculated using the Eqs. 5–8 as shown below: [18, 19]:
W¼
-23.81 -54.91
-107.84
-22.73 -21.65
would be available indicating that higher dose of STV is required for effective therapeutic action. Therefore, the development of drugs that do not bind significantly to (plasma) proteins becomes the important objective in the anti-HIV therapy and management [8]. Binding parameters The binding constant (K) and the number of ligands binding to protein (n) were calculated using the equation indicated below: logðFo F Þ=F ¼ logK þ nlog½Q
The values of K and n at 288, 298 and 308 K are listed in Table 1. The fact that the binding constant of STV– protein decreased with increase in temperature indicating that the stability of STV–HHb and STV–HSA complexes was weakened at higher temperatures. Further, the values of n close to unity indicated that one molecule of STV
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