DOI: 10.1002/asia.201201060

Unusual Binding of a Potential Biomarker with Human Serum Albumin

Dipanwita De, Harpreet Kaur, and Anindya Datta*[a]

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FULL PAPER

Introduction

Human serum albumin (HSA), the carrier protein in bloodplasma, acts as an internal delivery vehicle by binding sever-al biologically activated ligands, such as drugs, fatty acids,steroids, and surfactants.[1–4] HSA consists of a single poly-peptide chain that is made up of 585 amino acids with threea-helical domains (I–III). This structure can be further clas-sified into two subgroups, A and B (Scheme 1 a).[5] Hydro-

phobic pockets of almost the same size that are located insubdomains IIA and IIIA, namely, Sudlow’s site I and II, re-spectively, are the two primary high-affinity drug-bindingsites in HSA.[6–9] The crystal structures of the inclusion com-plexes of various drugs that were bound to HSA have beendetermined.[10–12] Such structural identification has shownthat bulky heterocyclic non-polar molecules, preferably witha centrally delocalized negative charge, are likely to bindspecifically to Sudlow’s site I (IIA). This type includes drugslike warfarin, azapropazone, paclitaxel, and phenylbutazoneand, thus, this site is also referred as a warfarin�azapropa-zone site.[13,14] On the other hand, drugs like ibuprofen,dansyl-l-proline, flufenamic acid, and diazepam, which con-tain a binding locus in Sudlow’s site II (IIIA) are character-ized by a localized negative charge on the a carbon of an ar-omatic compound that contains carboxylic-acid groups and,thus, this site is also known as an indole�benzodiazepinesite.[14] However, binding studies clearly demonstrate thatthese structural properties are not a prerequisite factor indetermining the binding locus of a drug in HSA. This resultholds true for drugs like aspirin and bilirubin, which are re-ported to bind to both sites I and II with different affini-ties.[13]

Conventional ligand-binding studies that consider the op-tical properties of HSA are generally performed by monitor-ing circular dichroism (CD) and fluorescence.[6,15,16] Biophys-ical techniques, such as isothermal titration calorimetry(ITC) and NMR-based screening methods, can also provideuseful information regarding the specific binding of drugs toproteins.[17,18] In spite of the complex structure of HSA, fluo-rescence measurements are possible, owing to the presenceof a lone tryptophan (Trp) group in subdomain IIA at posi-tion 214. The intrinsic fluorescence of Trp-214 is used to de-termine the binding location of a drug and it has found med-ical and biological applications in the design of variousdrug-delivery systems.[19–22] [2,2’-bipyridyl]-3,3’-diol(BP(OH)2), a fluorophore that exhibits excited-state intra-molecular double-proton transfer (ESIDPT), has been re-ported to be a potential biomarker, owing to its favorableinteractions with HSA.[23] Single-crystal X-ray analysis hasrevealed that the dienol tautomer (DE) of BP(OH)2 has

Abstract: This study investigates thespecific binding of a potential biomark-er, [2,2’-bipyridyl]-3,3’-diol (BP(OH)2),with human serum albumin (HSA).The binding of BP(OH)2 at the two pri-mary drug-binding sites on HSA (Su-dlow’s sites I and II) is explored bya competitive-binding study and moni-tored by considering the green-lightemission from its diketo tautomer.Warfarin is used as a marker for site Iand dansyl-l-proline (DP) as a competi-tor for site II. Steady-state and time-re-solved fluorescence measurements

affirm that neither of Sudlow’s sites isthe binding locus of BP(OH)2. To gainan idea regarding the probable bindingsite of BP(OH)2, we perform molecu-lar-docking studies, which reveala close proximity of the probe to Trp-214 in subdomain IIA of HSA. Confir-mation of this contention is achievedby studying the quenching of the fluo-

rescence of Trp-214 in the presence ofBP(OH)2. Moreover, static quenchingseems to be responsible for the deple-tion of the fluorescence of Trp-214, asmanifested by the invariance of the in-trinsic fluorescence lifetime of Trp-214,as a function of the concentration ofBP(OH)2. Based on displacement andquenching studies, supported by molec-ular docking, we propose thatBP(OH)2 binds in a cleft that separatessubdomains IIIA and IIB, which is inclose proximity to Trp-214.

Keywords: albumin · biaryls ·competition experiments · fluores-cence · interfaces

[a] D. De, H. Kaur, Prof. A. DattaDepartment of ChemistryIndian Institute of Technology BombayPowai, Mumbai 400 076 (India)Fax: (+91) 22-2570-3480E-mail : anindya@chem.iitb.ac.in

Supporting information for this article is available on the WWWunder http://dx.doi.org/10.1002/asia.201201060.

Scheme 1. a) Structure of HAS, with its six subdomains marked in differ-ent colors: IA green, IB yellow, IIA red, IIB magenta, IIIA blue, IIIBpurple; PDB ID: 1A2. b) Molecular structures of the different tautomersof BP(OH)2.

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a planar geometry.[24] The excited-state photophysics of thisprobe have been well-studied by using a femtosecond fluo-rescence upconversion techniques[25–27] and by transient-ab-sorption measurements.[28] The monoketo tautomer (MK),which is only formed in the excited state during stepwiseproton-transfer reaction, has a lifetime of 100 fs[29] and sub-sequently relaxes into its emissive diketo tautomer (DK) inabout 10 ps (Scheme 1 b).[30] Conversely, the concertedproton-transfer reaction has been reported to be ultrafast(less than 100 fs).[26]

The tautomerization process of BP(OH)2 in neat solventsprimarily involves two forms (Scheme 1 b).[31] The DE tauto-mer, which is associated with two intramolecular hydrogenbonds, exists in the ground state in all solvents and absorbsat 350 nm.[32] However, the DK that is generated duringESIDPT, is solely responsible for the strong green emissionin BP(OH)2, with a quantum yield that varies from 0.2 to0.4.[32] The ground state of the DK tautomer is only ob-served to an appreciable extent in water, owing to the for-mation of intermolecular-hydrogen-bonded water com-plexes.[31] This result is manifested by the presence ofa second absorption band in water between 400–430 nm.Both the DE and DK tautomers have negligible dipole mo-ments, as established by various theoretical[33, 34] and electro-optical measurements.[35] Owing to the nonpolar nature ofthe ground and excited states, this fluorophore has a propen-sity to bind to hydrophobic nanocavities in systems like cy-clodextrins,[23, 36] micelles,[31] zeolites,[37] binary mixtures of1,4-dioxane/water,[38] and HSA.[23] The encapsulation ofBP(OH)2 in albumin�SDS (sodium dodecyl sulfate) aggre-gates, as observed in our very recent study, can be used asa marker for determining the nature of protein–surfactantinteractions.[39] The binding site of BP(OH)2 in HSA has sofar been unexplored, although, based on its structural prop-erties, it is thought to bind in subdomain IIA.[23] Herein, weextend this work towards an experimental determination ofthe location of BP(OH)2 in HSA by using a competitive-binding technique.[1,14] Warfarin is used as a marker for site Iand dansyl-l-proline is used as a competitor for site II.These experimental results are supported by computationalmolecular-docking experiments.[40]

Results and Discussion

A brief discussion on the spectroscopic and temporal prop-erties of the free and HSA-bound complexes of the probe isrequired to better appreciate the experimental results. Onemay recall that the absorption spectra of BP(OH)2 in aque-ous buffer at pH 7 has two bands: one at 350 nm (which isascribed to the DE form) and another between 400–430 nm(the DK tautomer), as discussed previously.[31] In aqueoussolution, free BP(OH)2 emits in the green-light region at465 nm, with a fluorescence lifetime of 600 ps. Upon bindingto HSA, an increase in the emission intensity is accompa-nied by a red shift of 25 nm and an increase in the contribu-tion of the lifetime of the HSA-bound probe (to about

3.7 ns) as a function of HSA concentration, thus demon-strating the incorporation of the probe into the hydrophobicpockets of the protein.[23,39] A marked red shift in the emis-sion spectra, along with an increase in the lifetime ofBP(OH)2 from 600 ps in water to about 3.7 ns in HSA, indi-cate a stabilization of the excited state of the DK form inthe presence of HSA, most likely owing to shielding fromwater. For a competitive-binding study, the concentration ofHSA is chosen on the basis of two conditions: 1) a concen-tration of HSA that is much lower than that of BP(OH)2

and 2) a concentration of HSA that is higher than that ofBP(OH)2. Therefore, based on our previous study, the con-centrations of HSA that we chose for these displacementstudies were 5 mm and 40 mm.[39] The concentration of thecompetitor drug was varied between 0 and 44 mm. Impor-tantly, BP(OH)2 does not alter the secondary structure ofHSA, as revealed by the circular dichroism (CD) spectra re-cently reported by ourselves.[39]

Warfarin as a Marker for Sudlow’s Site I

Warfarin absorbs at around 305 nm in neat aqueous solution(pH 7), with an emission maximum at 390 nm, which is closeto the values reported earlier (see the Supporting Informa-tion, Figures S1 and S2).[1,41] The formation of its complexwith HSA is marked by small spectroscopic shifts, as report-ed elsewhere.[41,42] The binding constant of warfarin to Su-dlow’s site I in HSA, as reported in the literature, rangesfrom 2 �105 to 5 � 105

m�1.[41] The fluorophore under investi-

gation, namely, BP(OH)2, is known to form a 1:1 complexwith HSA, with an association constant of 5(�1) �104

m�1.[23]

Prior knowledge of the binding constants of the two HSAcomplexes that are formed with the warfarin marker andwith the BP(OH)2 probe is essential for a conclusive com-petitive-binding study. Because the displacement reaction iscontrolled by the values of the individual binding constants,it is expected that warfarin should displace BP(OH)2 fromits HSA complex because it has a higher binding constant.During the competitive-binding experiment, the fluores-cence intensity of BP(OH)2 is monitored because it is freefrom interference of the emission of HSA and bound war-farin. The emission maximum of HSA is at around 350 nmand that of bound warfarin is at 380 nm.[42]

The absorption spectrum of BP(OH)2 does not show anyappreciable change with increasing warfarin concentrationfrom 0 to 44 mm (Figure 1 a). The small increase in absorp-tion upon the addition of warfarin is probably due to thechange in the microenvironment that occurs in the presenceof warfarin. This result holds true for the emission spectraof BP(OH)2 at two different excitation wavelengths (350and 375 nm) in the presence of 0 and 44 mm warfarin (Fig-ure 2 a). Furthermore, the release of BP(OH)2 in aqueoussolution during the displacement reaction is expected to beaccompanied by a blue shift of the emission spectra, whichis not observed in the presence of the warfarin marker. Thisresult indicates that the binding locus of BP(OH)2 is not insubdomain IIA, where BP(OH)2 may be predicted to bind

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on the basis of the structural similarity of the probe withdrugs that are known to bind at the same site.[6] As men-tioned above, the structure of a drug cannot be the lone pa-rameter for determining its binding site in HSA.[43] Thus,time-resolved studies at lex =375 nm are carried out withthe aim of verifying the steady-state observations (Figure 3).

In the presence of both 5 and 40 mm HSA, BP(OH)2 showsa biexponential decay (Table 1) with a fast component(0.52–0.54 ns, assigned to the free probe in water) anda slow component (3.14–3.65 ns, assigned to the HSA-boundprobe). The contribution of the protein-bound BP(OH)2

complex increases as a function of HSA concentration, inline with the observed red shift in the emission spectra.[39]

Successive addition of warfarin is expected to result in a pro-gressively faster decay, owing to the discharge of BP(OH)2

in an aqueous environment by the marker. However, thepresence of warfarin does not alter the decay of BP(OH)2 at480 nm. This result corroborates with the steady-state obser-vations, thus strongly reinforcing the fact that BP(OH)2

does not bind to Sudlow’s site I. Therefore, in the next step,the possibility of its binding with Sudlow’s site II is ex-plored.

Dansyl-l-proline (DP) as a Marker for Sudlow’s Site II

In water, DP absorbs at 330 nm and emits at about 570 nm,which has a small overlap with the emission spectra ofBP(OH)2 (see the Supporting Information, Figures S1 andS2).[44] It may be useful here to mention that, although theemission of free DP is much more red-shifted than that ofBP(OH)2, the emission spectra of HSA-bound DP showsconsiderable overlap with the spectra of both free andHSA-bound BP(OH)2 (see the Supporting Information, Fig-ure S2). However, this study demonstrates the strength ofthe fluorescence-lifetime-measurement technique in resolv-ing such complex situations. Other molecules could alsohave been used; for example, ibuprofen has spectroscopicproperties that are very different to those of BP(OH)2 andit binds to Sudlow’s site II. However, it is reported to havea secondary binding site at the interface between subdo-mains IIA and IIB in HSA[45] and, hence, it is not used asa marker for this study. The association constant of DP withHSA (2 � 105

m�1), like that of warfarin, is one order of mag-

nitude higher than that of BP(OH)2.[46] Therefore, the dis-

placement measurements were performed by the successiveaddition of DP to a solution of BP(OH)2 in HSA, at thesame concentrations of the probe, protein, and the markeras before. The addition of DP to the solution of the proteinsolubilized by BP(OH)2 does not modify the absorptionspectra of the probe to any appreciable extent (Figure 1). Incontrast, the area under the O.D. (optical density)-correctedemission spectra, as a measure of the relative quantumyield, increases significantly upon excitation at both 350 and375 nm (Figure 2 b). The displacement of BP(OH)2 by DP is

Figure 1. Absorption spectra of BP(OH)2 in 5 and 40 mm HSA with thelowest (0 mm, solid line) and highest concentrations (44 mm, dashed line)of both markers, warfarin (WF) and dansyl-l-proline (DP); the concen-trations of the markers and HSA for each set of solutions are shown inthe legend. The absorption spectra of BP(OH)2 was obtained by subtract-ing the absorption spectra of the solubilized marker in HSA from thespectra of BP(OH)2 at the same concentrations of the marker and HSA.

Figure 2. O.D.-corrected emission spectra of BP(OH)2 in 5 and 40 mm

HSA with the lowest (0 mm, solid line) and highest concentration (44 mm,dashed line) of a) warfarin (WF) and b) dansyl-l-proline (DP) at lex =

350 nm and 375 nm; the concentration of HSA and the excitation wave-lengths are shown in the legend.

Figure 3. Fluorescence decay of BP(OH)2 in a) 5 mm HSA and b) 40 mm

HSA with increasing concentration of warfarin (WF) from 0 to 44 mm atlex =375 nm and lem =480 nm.

Table 1. Time-resolved fluorescence-decay parameters of BP(OH)2 in5 mm and 40 mm HSA with different concentrations of warfarin (WF) atlex =375 nm and lem =480 nm (pH 7).ACHTUNGTRENNUNG[HSA] [mM] [WF] [mM] t1 [ns] A1 t2 [ns] A2 c2

5 0 0.52 0.96 3.14 0.04 1.0944 0.51 0.96 3.07 0.04 1.17

40 0 0.54 0.89 3.65 0.11 1.0444 0.56 0.89 3.72 0.11 1.12

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expected to suppress the fluorescence quantum yield of theprobe.[31] However, it is difficult to comment on the proba-bility of binding BP(OH)2 in subdomain IIIA from thesteady-state data alone, owing to a considerable contributionfrom the fluorescence signal of the HSA-bound DP complexat 480 nm (see the Supporting Information, Figure S2).[46] Toresolve this complex situation, time-resolved fluorescencestudies on the solubilized probe and the marker in HSAwere performed at lex =375 nm (Figure 4). The fluorescence

decay became progressively slower with increasing DP con-centration from 0 to 44 mm. It should be remembered thatthis decay contains contributions from three different spe-cies: Free BP(OH)2, bound BP(OH)2, and DP. Protein-bound DP is reported to have a very long lifetime of20 ns;[44] for comparison, free BP(OH)2 has a lifetime of0.60 ns and HSA-bound BP(OH)2 has a lifetime of 3.7 ns.[39]

The fluorescence intensity of free DP at 480 nm is negligi-ble. Thus, it does not contribute to this decay to any appreci-able extent. With these considerations in mind, a globalanalysis of the fluorescence decay of BP(OH)2 and DP in 5and 40 mm HSA was performed. Triexponential functionswere used to fit the decays, with the lifetimes as global pa-rameters and amplitudes as local parameters. The three life-times that were obtained were easily assigned to freeBP(OH)2 in water (about 0.6 ns), the HSA bound-BP(OH)2

complex (about 4.0 ns), and the HSA-bound DP complex(about 20 ns; Table 2). The agreement of these lifetimeswith those reported earlier lends credibility to the soundnessof this global analysis. The amplitude (A3) of zero that wasobtained for the longest component further strengthens thecontention that this component should be assigned to pro-tein-bound DP. The increase in the A3 value with increasingconcentration of DP is in line with this contention as well.To ascertain whether DP displaces BP(OH)2 from its com-plexes with HSA or not, it is imperative to normalize thevalue of A1 or A2 with respect to their sum, so as to elimi-nate the increasing contribution from HSA-bound DP. Theparameter A1/ ACHTUNGTRENNUNG(A1+A2) denotes the fraction of freeBP(OH)2. This parameter remains invariant at all concentra-tions of DP, thus implying that DP does not displace

BP(OH)2 from its complex with HSA. Therefore, it may besaid with confidence that BP(OH)2 does not bind to Su-dlow’s site II either. Thus, to obtain an idea of where it doesbind, molecular-docking studies were performed.

Molecular-Docking Experiments

A blind docking study of BP(OH)2 to HSA suggests that themost-favorable binding site of the probe is located insidea hydrophobic pocket that is provided by a cleft at the inter-face of subdomains IIIA and IIB (Figure 5 a). The residues

Figure 4. Fluorescence decay of BP(OH)2 in a) 5 mm HSA and b) 40 mm

HSA with increasing concentration of dansyl-l-proline from 0 to 44 mm atlex =375 nm and lem =480 nm.

Table 2. Time-resolved decay parameters of BP(OH)2 in 5 mm and 40 mm

HSA with different concentrations of dansyl-l-proline (DP) at lex =

375 nm and lem =480 nm (pH 7).[a]ACHTUNGTRENNUNG[HSA][mM]

[DP][mM]

t1

[ns]A1 t2

[ns]A2 t3

[ns]A3 A1/ ACHTUNGTRENNUNG(A1+A2) c2

5 0 0.55 0.97 4.2 0.03 20.0 0.00 0.97 1.207 0.55 0.96 4.2 0.02 20.0 0.02 0.98 1.03

20 0.55 0.95 4.2 0.01 20.0 0.04 0.99 1.0234 0.55 0.92 4.2 0.01 20.0 0.07 0.99 1.0044 0.55 0.91 4.2 0.01 20.0 0.08 0.99 1.00

40 0 0.58 0.88 4.0 0.12 20.5 0.00 0.88 1.047 0.58 0.79 4.0 0.10 20.5 0.11 0.88 1.02

20 0.58 0.62 4.0 0.10 20.5 0.21 0.86 1.0034 0.58 0.66 4.0 0.09 20.5 0.25 0.88 1.0044 0.58 0.69 4.0 0.09 20.5 0.28 0.88 1.01

[a] The temporal parameters were obtained by using global analysis, inwhich the lifetimes were global parameters.

Figure 5. a) Minimum-energy conformation of the binding of BP(OH)2 inthe cleft that separates subdomains IIIA and IIB of HSA (PDB ID:1A2), as suggested by the molecular-docking study. b) A close view ofthe residues in the immediate vicinity of BP(OH)2 in the binding pocket.Distance [�] between the probe and the lone Trp-214 group that is locat-ed in subdomain IIA and the hydrogen-bond distance between the atompairs (side-chain hydrogen atoms of Arg-348 and Arg-485 and the hy-droxy oxygen atom of BP(OH)2) are marked in (b).

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in the immediate vicinity of BP(OH)2 in its binding pocketare Val-344 and Arg-348 from subdomain IIB and Pro-384,Met-446, Ala-449, Glu-450, Leu-453, and Arg-485 from sub-domain IIIA (Figure 5 b). Residues like Val, Pro, Ala, andLeu offer a hydrophobic microenvironment, which presuma-bly facilitate the anchoring of the non-polar probe insidethis pocket. The most-favorable docking mode of boundBP(OH)2 exhibits the formation of bifurcated hydrogenbonds between one of the hydroxy oxygen atoms ofBP(OH)2 and the side-chain hydrogen atoms of Arg-348and Arg-485. The formation of such hydrogen bonds withamino-acid residues in the protein is known to stabilize thebound form of other drugs.[47,48] Docking studies displaya close proximity between BP(OH)2 and the Trp-214 residuein the HSA-bound complex. The two moieties are separatedby a distance of about 11 � (Figure 5 b), with a binding freeenergy change of �5.27 kcal mol�1. Similar studies on thepossibility of binding to cleft regions near Trp-214 by otherfluorophores have been reported in the literature.[49] Withthe aim of validating the proximity of BP(OH)2 to the lonetryptophan residue (Trp-214), as shown by docking studies,the fluorescence of Trp-214 was monitored at lex =295 nm.

Fluorescence Quenching of HSA in the Presence ofBP(OH)2

On the subsequent addition of BP(OH)2 from 0 to 18 mm,the emission spectra show a progressive quenching of thefluorescence of Trp-214 at 350 nm (Figure 6). This quench-

ing is accompanied by the onset of an increase in the emis-sion of the BP(OH)2 probe. The presence of an isoemissivepoint in the emission spectra reinforces the conclusion thata two-state equilibrium process is operative. To explorewhether the quenching is static or dynamic in nature, time-resolved fluorescence decays are recorded by exciting Trp-214 at 295 nm. The evolution of HSA over time remains un-affected by the addition of the probe, thus signifying thatstatic quenching is solely responsible for the depletion ofthe fluorescence of Trp-214 (see the Supporting Information,Figure S3).[50] Moreover, the possibility of quenching owingto FRET is ruled out here, because the decay of the donor

(Trp-214) remains unaltered with the subsequent addition ofan acceptor (BP(OH)2). A rather simplistic attempt wasmade to evaluate the association constant of the HSA-bound BP(OH)2 complex by using the well-known Stern–Volmer equation for static quenching (Equation (1)), whereF0 and F are the fluorescence intensity of HSA in the ab-sence and presence of a quencher (Q, BP(OH)2), respective-ly, [Q] is the concentration of the quencher, and KS, theStern–Volmer constant, is essentially the association con-stant.

F0

F� 1 ¼ KS½Q� ð1Þ

A linear Stern–Volmer plot is obtained, thus indicatingthat a static quenching mechanism operates in this system(Figure 6, inset).[50] The KS value as determined from theslope is 3.8 � 104

m�1, which corroborates with previously re-

ported values for this complex.[23] Therefore, the obtained Ks

value is used to calculate the change in binding free energy,which is defined as in Equation (2).

DG0binding ¼ �2:303RT log KS ð2Þ

The value of DG0binding is �6.21 kcal mol�1, which is close

to the free-energy change that was obtained from the dock-ing study. Furthermore, quenching of the fluorescence ofHSA indicates that BP(OH)2 is indeed in close proximity toTrp-214 in its complex with the protein.

Conclusions

The binding site of BP(OH)2 in HSA has been ascertained.Competitive-binding studies with two known markers, war-farin and dansyl-l-proline (DP), at the principle drug-bind-ing sites, namely, Sudlow’s sites I and II (located in subdo-mains IIA and IIIA, respectively), have been performed.Owing to the higher binding constants of the HSA-boundmarker complexes, warfarin and DP were added separatelyto a solution that contained a HSA-bound BP(OH)2 com-plex and its green emission was monitored by using steady-state and time-resolved measurements. The green emissionremained unaffected by the presence of either of these twomarkers, thereby eliminating the possibility of BP(OH)2

binding to the pockets in subdomain IIA or IIIA. The prob-able location of BP(OH)2 within HSA was determined bymolecular-docking experiments, from which a cleft at the in-terface of IIIA and IIB seemed to be the minimum-energybinding site for the probe. In addition to stabilization fac-tors, like hydrophobicity and hydrogen-bond interactionswith the residues in their immediate vicinity, the probe wasfound to be in close proximity to Trp-214 in the minimum-energy docking pose. Experimental support for this theorywas provided by quenching of the fluorescence of Trp-214by the addition of BP(OH)2 and this quenching mechanismwas primarily static in nature. The free-energy change of

Figure 6. Emission spectra of HSA at lex =295 nm with increasing con-centration of BP(OH)2 in a buffer solution at pH 7; arrows indicate an in-crease in the concentration of BP(OH)2 from 0 to 18 mm. Inset: Plot ofF0/F�1 as a function of [BP(OH)2] in the presence of 40 mm HSA.

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binding, as calculated by using quenching studies, was in linewith the computational results. Thus, in conjugation with ex-perimental displacement measurements and computationalmolecular-modeling studies, one may infer that none ofthese two primary drug-binding sites accommodate theprobe; rather, the cleft between subdomains IIIA and IIBseems to provide a favorable binding locus for BP(OH)2.

Experimental Section

Human serum albumin (HSA, fatty acid and globulin free, A3782), war-farin, and dansyl-l-proline were purchased from Sigma. Sodium dihydro-gen phosphate (guaranteed reagent (GR) grade) and disodium hydrogenphosphate (GR grade), which were used to prepare the buffer solution(pH 7), were purchased from Merck. [2,2’-bipyridyl]-3,3’-diol [BP(OH)2](98 %) was purchased from Aldrich. All of the compounds were used asreceived without any further purification. All measurements were per-formed in phosphate buffer (pH 7) with an ionic strength of 0.2. The con-centration of BP(OH)2 for the competitive-binding studies was adjustedto 18 mm. Absorption and emission spectra were recorded on a JASCOV-530 spectrophotometer and a Varian Cary Eclipse spectrofluorimeter,respectively. The emission spectra of BP(OH)2 and HSA were recordedat excitation wavelengths of 350 nm, 375 nm, and 295 nm, with both exci-tation and emission slits fixed at 5 nm. A picosecond-pulsed diode-laser-based time-correlated single-photon counting (TCSPC) instrument,which was set at a magic angle of 54.78 (IBH, United Kingdom) was usedto record the time-resolved fluorescence decay. Samples were excited at375 nm (laser) and 295 nm (LED) with FWHMs of 250 and 750 ps, re-spectively. Multiexponential fitting of the decay traces was performed byusing IBH DAS 6.2 data-analysis software.[51, 52]

The structure of BP(OH)2 was geometrically optimized at the 6-31G*level by employing the Becke three-parameter Lee–Yang–Parr (B3LYP)hybrid density functional theory with the Gaussian 09[53] software pack-age, which converged into a planar structure, as obtained by single-crystalX-ray analysis.[24] The resulting energy-minimized structure was used toperform the molecular-docking experiments of BP(OH)2 to the HSA pro-tein crystal structure (PDB ID: 1A2), by using the Autodock 4.2 softwarepackage.[54] All of the torsional angles in BP(OH)2 were kept rigid tomaintain the co-planarity of the two pyridyl rings. Gasteiger partialcharges were added to the ligand atoms and non-polar hydrogen atomswere merged by using Autodock tools. All of the water molecules in thecrystal were removed, polar hydrogen atoms were added, and Kollmanunited-atom partial charges[55] were assigned to the HSA protein.

Blind docking of BP(OH)2 to HSA was performed by keeping the dimen-sions of the gridbox sufficiently large, that is (126, 76, 126) grid points, toplace the whole protein at its center (28.6, 9.527, 19.336), with a gridspacing of 0.63 �. By applying the Larmarckian genetic algorithm,[56]

200 individual docking runs were carried out. Starting with an initial pop-ulation of 150 randomly placed individuals, a maximum number of 2.5�107 energy evaluations, a mutation rate of 0.02, a crossover rate of 0.8,and an elitism value of 1, the worst individual in the current populationwas determined by taking an average over a window of the 10 precedinggenerations into consideration. The Solis and Wets algorithm[57] was im-plemented to perform the local search, with a maximum of 300 iterations.The probability of subjecting an individual in the population to a localsearch was 0.06 and the maximum number of consecutive successes orfailures before doubling or halving the local search step size was 4. Theresulting structures were divided into different clusters with a root-mean-square tolerance of 2 �. Then, these clusters were ranked on the basis ofthe mean binding energy of the structures in each cluster. The structurethat possessed the lowest binding energy in the cluster, ranked first, wasthen selected as the preferred mode of ligand binding to the protein. Thisstructure was then analyzed by using VMD.[58]

Acknowledgements

This work was supported by the SERC and the DST. D.D. thanks theCSIR for a Senior Research Fellowship.

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38.Received: November 7, 2012

Published online: February 12, 2013

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