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Toxicology 292 (2012) 78– 89

Contents lists available at SciVerse ScienceDirect

Toxicology

j ourna l ho me pag e: www.elsev ier .com/ locate / tox ico l

igarette smoke induces p-benzoquinone–albumin adduct in blood serum:mplications on structure and ligand binding properties

runava Ghosha, Aparajita Choudhurya, Archita Dasa, Nabendu S. Chatterjeeb, Tanusree Dasc,ukhsana Chowdhuryc, Koustubh Pandaa, Rajat Banerjeea,∗∗, Indu B. Chatterjeea,∗

Department of Biotechnology and Dr. B. C. Guha Centre for Genetic Engineering & Biotechnology, Calcutta University College of Science, 35, Ballygunge Circular Road, Kolkata00019, IndiaNational Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road, Scheme-XM, Beliaghata, Kolkata 700010, IndiaIndian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 700032, India

r t i c l e i n f o

rticle history:eceived 12 October 2011eceived in revised form 31 October 2011ccepted 22 November 2011vailable online 1 December 2011

eywords:uman serum albuminigarette smoke-BenzoquinoneALDI-MS

a b s t r a c t

Earlier we had reported that irrespective of the source cigarette smoke (CS) contains substantial amountsof p-benzosemiquinone, which is readily converted to p-benzoquinone (p-BQ) by disproportionation andoxidation by transition metal containing proteins. Here we show that after CS-exposure, p-BQ-proteinadducts are formed in the lungs as well as serum albumin of guinea pigs. We also show that serum ofhuman smokers contains p-BQ-albumin adduct. It is known that human serum albumin (HSA) plays avery important role in binding and transport of a variety of ligands, including fatty acids and drugs. Weshow in vitro that p-BQ forms covalent adducts with free amino groups of all twenty amino acids as well as�-amino groups of lysine residues of HSA in a concentration dependent manner. When HSA is incubatedwith p-BQ in the molar ratio of 1:1, the number of p-BQ incorporated is 1. At the molar ratio of 1:60,the number of p-BQ incorporated is 40. The formation of HSA–p-BQ adduct has been demonstrated by

luorescence spectroscopyigand binding

absorption spectroscopy, MALDI-MS and MALDI-TOF–TOF-MS analyses. Upon complexation with p-BQ,the secondary structure and conformation of HSA are altered, as evidenced by steady state and time-resolved fluorescence, circular dichroism, 8-anilino-1-napthalenesulfonic acid binding and differentialscanning calorimetry. Alteration of the structure and conformation of HSA results in impairment of itsligand binding properties with respect to myristic acid, quercitin and paracetamol. This might be one ofthe reasons why transport and distribution of lipids and drugs are impaired in smokers.

. Introduction

Numerous scientific and epidemiological evidences havenequivocally established that cigarette smoking is a risk factoror various degenerative diseases like cancer of the lungs andther organs (Bartecchi et al., 1994; Calle et al., 1994; Giovino,002; Kuper et al., 2002; Boffetta, 2008; Sequist, 2008), emphy-ema (Lopez and Murray, 1998; Tuder et al., 2003; Banerjee

t al., 2008), and cardiovascular disease (Services, 1989). Even inmokers without any apparent clinical symptoms, the overall lifexpectancy is reduced, ranging up to 10 years fewer than non-

Abbreviations: HSA, human serum albumin; CS, cigarette smoke; AECS, aqueousxtract of cigarette smoke; p-BSQ, p-benzosemiquinone; p-BQ, para-benzoquinone;SC, differential scanning calorimetry; ANS, 8-anilino-1-naphthalenesulfonic acid.∗ Corresponding author. Tel.: +91 33 24615445x393; fax: +91 33 24614849.

∗∗ Corresponding author. Tel.: +91 33 24615445x394; fax: +91 33 24614849.E-mail addresses: rbbcgc@gmail.com (R. Banerjee), ibc123@rediffmail.com

I.B. Chatterjee).

300-483X/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.tox.2011.11.014

© 2011 Elsevier Ireland Ltd. All rights reserved.

smokers (Doll et al., 2004). One reason that had been consideredfor the pathogenesis of most of the degenerative diseases is oxida-tive damage of proteins, lipids and DNA (Cross et al., 1993; Asamiet al., 1997; Santanam et al., 1997). The oxidants present mainlyin the tar phase (Panda et al., 2001) had been identified to bestable water soluble semiquinone radicals, particularly, o- and p-benzosemiquinones (Pryor et al., 1983; Pryor et al., 1998). Thewater soluble oxidants of CS can readily reach both the systemiccirculation (Csiszar et al., 2008) and interstitial cells (Ishii et al.,2001) leading to various degenerative diseases. Subsequently, weisolated the stable oxidants from aqueous extract of CS (AECS)and characterized it as p-benzosemiquinone (p-BSQ) (Banerjeeet al., 2008). We have observed that the mainstream smoke fromall commercial cigarettes examined as well as Kentucky researchcigarettes contains substantial amounts (100–200 �g/cigarette) ofp-BSQ (Chatterjee, 2005; Dey et al., 2010). Under physiological

conditions, p-BSQ is readily converted to p-BQ by disproportion-ation (Sullivan and Reynolds, 1996) and oxidation by transitionmetal containing proteins (Banerjee et al., 2008). p-BQ is a redoxcycling agent that produces reactive oxygen species (ROS) leading

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o oxidation of proteins and DNA (Bolton et al., 2000). Although CSoes not contain p-BQ, it is conceivably produced in the smoker’s

ungs through oxidation of p-BSQ by Cu, Zn-SOD and cytochrome (Banerjee et al., 2008). Recently, we have reported that AECS-nduced formation of ROS and oxidative damage in human lungells (A549) is not only completely prevented by antibody to p-Q, but also mimicked by p-BQ in amounts present in AECS (Deyt al., 2011). This indicates that the causative factor responsible forECS-induced oxidative damage is p-BQ.

However, besides being a redox cycling agent generating ROS, p-Q functions as a strong arylating agent producing Michael adductsith proteins (Bolton et al., 2000). Earlier we had shown that p-Q forms covalent adducts with BSA (Banerjee et al., 2008). Wead also shown that p-BQ forms protein adducts in the bone mar-ow of CS-exposed guinea pigs (Das et al., 2011). This indicates that-BQ formed in the lungs, gets into the blood stream, and theno different tissues of the body. Thus it would thus be interest-ng to investigate whether p-BQ interacts with serum albumin inmoker’s blood.

Human serum albumin (HSA) is the principal extracellular pro-ein of blood plasma accounting for about 60% of the total proteinHe and Carter, 1992; Dockal et al., 1999). HSA plays a very impor-ant role in binding and transport of an unusually broad spectrumf exogenous and endogenous ligands, including fatty acids and aariety of drugs (Bhattacharya et al., 2000; Ghuman et al., 2005;arshney et al., 2010). The distribution, free concentration and

ransport of various ligands significantly depend on their binding toSA (Peters, 1996). It is conceivable that any alteration of the struc-

ure and conformation of HSA by some xenobiotic might result inmpairment of binding and transport of essential ligands, affectingheir overall distribution and efficacy. As mentioned earlier, oneuch xenobiotic is p-BQ, which is derived most commonly fromxposure to CS (Banerjee et al., 2008; Das et al., 2011; Dey et al.,011).

Since p-BQ forms covalent adducts with BSA (Banerjee et al.,008), it would conceivably form adduct also with HSA. It is possi-le that in smokers, p-BQ forms covalent adduct with HSA resulting

n alteration of structure and impairment of ligand binding, includ-ng lipid, which is the primary physiological ligand of the proteinBhattacharya et al., 2000). p-BQ mediated alteration in serum lipidransport may be one of the underlying mechanisms for accumu-ation of lipids in serum of smokers. It is reported that smokersave significantly higher serum cholesterol, triglyceride, and low-ensity lipoprotein levels (Craig et al., 1989).

HSA has long been the centre of attention of also the phar-aceutical industries. The efficacy of a drug depends on the

inding with HSA, transport, distribution and the pharmacokineticroperty that means whether the drug is getting to its site ofction. Like that envisaged with lipids, p-BQ-induced alteration ofSA structure may also impair the pharmacokinetic properties ofrugs.

However, it is yet to be known whether CS exposure actuallyroduces the HSA–p-BQ adduct in vivo and whether such an adductesults in alteration of structure and impairment of ligand bindingroperties, including lipids and drugs. Here we show that serumf smoker’s blood contains HSA–p-BQ adduct. We demonstrate initro that p-BQ forms covalent adducts with �-amino groups of Lysesidues of HSA in a concentration dependent manner leading tolteration of HSA’s secondary structure and conformation result-ng in impairment of binding with myristic acid. HSA is also knowno play an important role in binding and transport of quercitin, aaturally occurring flavonoid having antioxidant and cardioprotec-

ive activity as well as paracetamol, a commonly used analgesic andntipyretic drug (Manach et al., 1995; Kaldas et al., 2005; Daneshgart al., 2009). We further show that bindings of quercitin and parac-tamol are altered upon complexation with p-BQ.

292 (2012) 78– 89 79

2. Materials and methods

2.1. Material

HSA was purchased from Sigma [A-1887 fatty acid free] and used without fur-ther purification. p-Benzoquinone (p-BQ) was obtained from Himedia [RM-489]and freshly recrystallized from n-hexane before use. All other reagents used wereof AR grade. All the spectrofluorimetric analyses were carried out in a Hitachi F-7000 spectrofluorimeter using a 10 mm or 5 mm path length (as needed) quartzcell. Absorbance spectra were taken in a Shimadzu UV-2540 spectrophotometer.

2.2. Ethics statement

All methods were approved by the Institutional Animal Ethics Committee, Per-mit No. 797/CPCSEA, University of Calcutta. All efforts were made to minimizesuffering of the animals. The collection of blood and subsequent experiments withhuman serum were approved by the Institutional Bioethics Committee for animaland human research studies, University of Calcutta, permission no. 1096, followingthe Code of Ethics of the World Medical Association (Declaration of Helsinki) forexperiments involving humans.

2.3. Collection of blood from human volunteers (smokers and non-smokers)

Written consents were obtained from all the volunteers prior to collection ofblood. All donors used in the study were male and in the age group of 25–35 years.Smokers used in this study had a smoking habit for 5 years. Nonsmokers used ascontrol had no prior history of smoking for the last 10 years. Venous blood was takenand serum was isolated.

2.4. Exposure of guinea pigs to cigarette smoke

The procedure was essentially same as that described before (Banerjee et al.,2008). Briefly, male short hair guinea pigs weighing 350–450 g were fed ascorbatefree diet for 7 days to minimize the vitamin C level of the tissues. This is becausevitamin C is a potential inhibitor of CS-induced tissue damage (Panda et al., 1999;Panda et al., 2000; Banerjee et al., 2008). After 7 days of vitamin C deprivation, theanimals were exposed to smoke from 5 Kentucky research cigarettes 3R4F/day @ 2puffs/cigarette. The total duration of exposure to smoke from one puff was 1 min 30 s,allowing the animal 1 min rest in smoke-free atmosphere to breathe air betweeneach puff. The gap between one cigarette and the next was 1 h. The procedure wasessentially same as that described before (Banerjee et al., 2008). The smoke chamberused was a vacuum desiccator (2.5 L) with an open tube at the top and a side tubefitted with a stopcock. The cigarette placed at the top was lit and CS was introducedinto the chamber containing the guinea pig by applying a mild suction through theside tube for 5 s. Thereafter, the vacuum was turned off and the guinea pig wasfurther exposed to the accumulated smoke for another 45 s. Pair-fed sham controlswere subjected to air exposure instead of CS under similar conditions. At the endof the experimental period, the guinea pigs were euthanized using i.p. injectionof ketamine hydrochloride (100 mg/kg guinea pig) and blood and lung tissue werecollected for further experiments.

2.5. Incubation of HSA with aqueous extract of CS (AECS) and p-BQ

Aqueous extract of CS (AECS) was prepared as described before (Panda et al.,1999). Briefly, smoke from one Kentucky research cigarette 3R4F was extracted with1 ml of 50 mM potassium phosphate buffer, pH 7.4, filtered through 0.22 �m Mil-lipore filter and the pH adjusted to 7.4. The method of preparation of AECS wasso devised as to simulate the manner in which the respiratory tract lining fluid isexposed to CS during the process of smoking by humans. The AECS solution thusobtained was used immediately. For experiments with AECS or p-BQ-treated HSA,HSA (1 mg) was incubated with 50 �l of AECS or 5 �g p-BQ in a final volume of 200 �l20 mM potassium phosphate buffer (pH 7.4).

2.6. Immunoblot analysis

Reaction mixtures containing 15 �g protein equivalent of HSA and lung tissuewere resolved on 10% SDS–PAGE and transferred to PVDF membrane (Santa CruzBiotechnology, inc.; sc-3723) and blocked with 5% non-fat dry milk (Himedia RM-1254). Primary antibody used was a double affinity purified polyclonal antibodyraised in rabbit against a conjugate of bovine serum albumin and p-BQ (supplied byAbexome Biosciences Pvt. Ltd., India). Blots were then incubated with goat anti rabbitIgG-HRP secondary antibody (GeNei, 105499). The membranes were finally detectedby chemiluminescence (LumiGLO Reagent and Peroxide, Cell Signaling Technology).

2.7. Identification of the mass of intact proteins by MALDI-MS

HSA was incubated with p-BQ at different molar ratios (HSA–p-BQ ≈ 1:1 to 1:60)in 20 mM ammonium bicarbonate buffer, pH 7.4, for 2 h at 37 ◦C and then dialyzedin the same buffer overnight. The dialyzed material was lyophilized. The lyophilizedsamples were dissolved in sample diluents (60% acetonitrile containing 0.1% TFA)

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o obtain a protein concentration of 1 mg/ml and 10 �l of this protein solution wasesalted by reverse-phase ZipTip C18 pipette tips (Millipore, USA) and applied to theALDI sample plate and mixed with equal volume of �-cyano-4-hydroxycinnamic

cid (CHCA) matrix directly on the target plate. Samples were allowed to dry foreveral minutes before MALDI-TOF MS measurements were performed. Spectraf intact protein masses were obtained on an MALDI-TOF/TOF mass spectrome-er (Model: 4800 MALDI-TOF–TOF, Applied Biosystems, USA). Mass spectra wereecorded in the m/z range between ∼20 and ∼200 kDa.

.8. MALDI-TOF–TOF-MS analysis of HSA–p-BQ adduct in smokers’ serum

Coomassie (R-250/Biosafe) stained protein bands of interest were cut manuallyrom SDS–PAGE gel and placed into microcentrifuge tubes. Proteins were digestedsing In-gel tryptic digestion kit (PIERCE) and processed according to the man-facturer’s instruction. Briefly, the digested peptides were dried in vacuum andubjected to ZipTipC18 purification for MS analysis. This was followed by iden-ification of proteins using a MALDI-TOF–TOF mass spectrometer (Model: 4800

ALDI-TOF–TOF, Applied Biosystems, USA) equipped with a special analysis soft-are GPS. All searches were performed against suitable databases.

.9. Spectrophotometry of p-BQ conjugates with amino acids and N-acetyl aminocids

Each of the common 20 amino acids and N-acetyl amino acids (500 nmol) wasncubated with or without 100 nmol of p-BQ in a final reaction volume of 200 �l forwo h at 37 ◦C in 20 mM potassium phosphate buffer, pH 7.4. The N-�-acetyl aminocids used were N-�-acetyl Ala, N-�-acetyl Cys, N-�-acetyl Trp, N-�-acetyl Arg and-�-acetyl His, N-�-acetyl Lys and N-�-amino Lys. Before incubation, the pH of themino acid solution of Lys, Arg, His, Asp and Glu was adjusted to 7.4. The reactionixture obtained after incubation of the respective amino acid with p-BQ as well

s the control was diluted to 1 ml by adding 800 �l of 20 mM potassium phosphateuffer, pH 7.4, and spectra were taken in the range of 200–700 nm.

.10. Estimation by free amino groups of HSA and HSA–p-BQ adducts byuorescamine fluorescence

After incubation of HSA with p-BQ in the molar ratio of 1:1 up to 1:60 asescribed above under MALDI-MS, the reaction mixture was added to 1.35 ml ofodium borate buffer (200 mM, pH 8.5) and mixed with 180 �l of fluorescamine1 mM in acetone). After incubation at room temperature for 15 min in the dark,uorescence was measured. Taking the fluorescamine fluorescence of unreactedSA at 474 nm as 100%, the decrease in fluorescence was measured (Colombo et al.,010).

.11. In-solution digestion and identification of peptide-p-BQ complex byALDI-TOF–TOF-MS

Ten microgram of HSA p-BQ adduct in the molar ratio of 1:1 was dissolvedn 15 �l of 50 mM ammonium bicarbonate (ambic) buffer and in-solution trypticigestions (200 ng trypsin, at 37 ◦C for overnight) were performed after the reduc-ion/alkylation of the samples using dithiothreitol (DTT: 100/200 mM at 95 ◦C for

min) and iodoacetamide (IAA: 200 mM at ambient temperature for 20 min in dark).he digested peptide fractions were vacuum-dried and dissolved in 10 �l of sampleiluent (60% acetonitrile containing 0.1% TFA) and desalted by reverse phase ZipTip18 pipette tips (Millipore, USA) prior to applying on to the MALDI sample plate. Amall fraction (0.4 �l) of the sample was spotted on the well first and equal volume of-cyano-4-hydroxycinnamic acid (CHCA) matrix was overlaid with a hydrophobicask on each spots and allowed to crystallize at room temperature. MALDI-TOF–TOFeasurements in the positive reflector mode were performed with an ABI 4800 Pro-

eomics Analyzer (Applied Biosystems, USA). The laser wavelength was 355 nm, andhe laser repetition rate was 200 Hz. The mass spectra were externally calibratedith the autodigest peaks of trypsin (MH+, 906.505, 1020.504, 1153.574, 2163.057

nd 2273.160 Da). The MS/MS data were acquired and processed using the GPS soft-are and MASCOT or SWISS-PROT was used to search the databases to identify theeptides. The following parameters were used in the searches: trypsin digest (oneissed cleavage allowed); mass accuracy 0.2 Da, and a mass tolerance of 50 ppm was

sed for the peptide search; taxonomy: Homo sapien; acetylation of the N-terminus,lkylation of cysteine by carbamidomethylation and oxidation of methionine wereonsidered as possible modifications.

MALDI-TOF–TOF measurements in the positive reflector mode were performedith an ABI 4800 Proteomics Analyzer (Applied Biosystems, USA).

.12. Fluorescence and absorption spectra of the HSA–p-BQ adduct

HSA (131 �mol) was incubated with or without different concentrations of p-

Q ranging from molar ratio of 1:1 to 1:60 in 20 mM potassium phosphate bufferpH 7.4) at 37 ◦C for 2 h. After incubation the reaction mixture was subjected tovernight dialysis against buffer at 4 ◦C to remove any free p-BQ or hydroquinoneHQ), a metabolite of p-BQ produced after interaction with HSA. The dialyzed reac-ion mixture was used for further studies. For time dependent absorbance spectrum

292 (2012) 78– 89

analysis, HSA was incubated with p-BQ (1:60) and small aliquots were drawn fromthe reaction mixture at different interval of time to prepare a 2.6 �M solution andabsorbance spectrum was acquired.

2.13. Fluorescence and absorption spectra of peptide-p-BQ complex

Each custom-made decapeptide (308 nmol) was incubated with 1540 nmol of p-BQ (in the molar ratio of 1:5) in 20 mM potassium phosphate buffer, pH 7.4, at 37 ◦Cin a final volume of 200 �l for 2 h. Controls were incubated separately using onlypeptide. Then the reaction mixture was extracted successively twice with n-butanol(200 �l each time) to remove any unreacted p-BQ or hydroquinone (produced fromp-BQ) present in the reaction mixture. The upper layer of butanol was discardedand the aqueous layer obtained after second butanol extraction was used for bothspectrophotometry and fluorescence study.

2.14. Fluorescence studies

Unless mentioned otherwise, all the fluorescence samples were excited at awavelength of 295 nm for selective excitation of tryptophan residues. The emissionspectra were recorded from 310 to 450 nm. The path length of the cuvette usedwas 0.5 mm to minimize the inner filter effect. For the same reason, the proteinconcentration used was 0.5 �M and the optical densities of the samples were keptbelow 0.1.

2.15. Fluorescence lifetime

Fluorescence lifetime measurements were determined from the total emissionintensity decays of 10 �M reaction mixtures, using a nanosecond time-domainfluorimeter and operated in the time-correlated single-photon-counting mode.Excitation was provided by a pulsed, high-pressure (1.5 atm) N2 lamp operatingat 25 kHz, the FWHM of the excitation pulse profile being 1.3 ns. The N2 emissionline at 297 nm was used to excite Trp, while the emission was monitored at 340 nm.Slits of bandwidth 16 nm were used in both excitation and emission channels. Meanfluorescence lifetimes were calculated using the relation 〈�〉 =

∑(˛i�2

i)/

∑(˛i�i),

where ˛i represents the fractional contribution to the time resolved decay of thecomponent having lifetime of � i the decay curve was fitted to three exponentialdecay with least chi-square value.

2.16. CD analysis

Reaction mixtures prepared for the measurements of fluorescence and absorp-tion spectra of HSA–p-BQ adducts, as described above, was diluted to 5 �M for farUV CD analysis and 10 �M for near UV CD analysis. CD spectra were recorded on aJasco J720 spectropolarimeter by scanning reaction mixtures from 200 to 250 nmusing 1 mm path length cuvette and 250 to 320 nm for near UV region using 10 mmcuvette path length. Mean residual ellipticity (MRE) was calculated using the for-mula, MRE = [100 × Observed(�)MW]/[10 × C × n × l] in deg cm2 dmol−1, where � isthe observed ellipticity in millidegree, MW is the molecular weight in kDa, C is theconcentration in mg/ml, n is the number of amino acids and l is the path length incm. The �-helix percentage was calculated using MRE value at 222 nm according tothe formula, [(MRE − 2340)/30300] × 100 (Chen et al., 1972)

2.17. ANS binding

To 1 �M reaction mixture containing HSA and HSA–p-BQ complex, ANS wasadded from a stock of 1 mM solution to achieve a final concentration of 10 �M.ANS binding was studied using 420 nm as the excitation wavelength and 482 nm asemission wavelength.

2.18. Differential scanning calorimetry (DSC) analysis

DSC measurements were performed using a nanocalorimeter, (N-DSC II,Calorimetry Sciences Corp, Utah U.S.A.). Pure, dialyzed samples of HSA–p-BQadducts or native HSA (3 mg/ml; 220.6 �M) in phosphate buffer (pH 7.4) were heatedfrom 25 to 100 ◦C at the rate of 1◦C/min increment using the same buffer as blank.DSC data were corrected for instrument baselines (determined by running the dial-ysis buffer in both reference and sample cells just prior to placing protein in thesample cell) and normalized for scan rate and protein concentration. Data analyseswere performed with Cpcalc software (Calorimetry Sciences Corp, Utah U.S.A.).

2.19. Ligand binding

2.19.1. Myristic acid

The stock solution of MA (1 mM) was prepared in 20 mM potassium phosphate

buffer, pH 7.4, by heating at 50 ◦C followed by cooling to room temperature. Myristicacid was added from the stock solution in 3 �l aliquots up to a final concentration of51–1 �M native HSA or HSA–p-BQ adduct. The excitation wavelength was 295 nmand the emission recorded at 340 nm.

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Fig. 1. Immunoblot analysis of lung proteins and blood serum of guinea pigs exposedto air or cigarette smoke using anti p-BQ antibody. Smoke exposure was made @ 5cigarettes/day for 14 days (6 days a week). (A) Lung tissue; (B) blood serum; 15 �gprotein was loaded in each case. Loading controls: �-tubulin in (A) and ponceau Sstaining of the membrane in (B). Details of the procedure are given under Section 2.

Fig. 2. (I) Immunoblot analysis of blood serum of human smokers and nonsmokersusing anti p-BQ antibody. Sex and age of nonsmokers and smokers: male, around 30years. Smokers No. 3 and 4: 5 cigarettes/day for 5 years (1.25 pack year); No. 5 and 6:10 cigarettes/day for 5 years (2.5 pack year). Lane 7 is pure HSA. Fifteen microgramprotein was loaded in each well. (II) ponceau S staining of the membrane as theloading control. Details of the procedure are given under Section 2.

Fig. 3. (I) Immunoblot analysis of HSA treated with and without aqueous extractof cigarette smoke (AECS) or p-BQ using anti p-BQ antibody. Fifteen microgram

A. Ghosh et al. / Tox

.19.2. QuercetinSince quercetin has substantial optical density at 295 and 340 nm that may cause

nner filter effect during titration by quenching method, we employed fluorescencenisotropy method which is suitable for measuring protein–ligand dissociationonstant and much less sensitive to inner filter effect than quenching. A 10 mMoncentrated stock solution of quercetin was prepared in ethanol. From this stockolution, further diluted stock solution of quercetin was prepared in buffer. In a finalolume of 500 �l, 5 �M quercetin was incubated with increasing concentrations ofrotein upto 50 �M. Amount of ethanol in the final incubation system was less than%.

.19.3. ParacetamolA 50 mM stock solution of paracetamol was prepared by dissolving paracetamol

n buffer. The concentration was determined using 10,236 M absorptivity of parac-tamol at 243 nm (Abdellatef et al., 2007). Small aliquots were added from dilutedaracetamol stock to a 0.5 �M free HSA or HSA–p-BQ complex upto a concentra-ion of 1.9 mM. The Tryptophan fluorescence quenching was measured using anxcitation wavelength of 295 nm and measuring emission at 340 nm.

.20. Determination of dissociation constant

.20.1. Anisotropy measurementsSteady-state anisotropy was recorded with a Hitachi model F-7000 spectrofluo-

ometer equipped with a polarization accessory. Excitation of quercetin was done at00 nm. The fluorescence anisotropy (A) values were obtained using the expression

= (IVV − GIVH)/(IVV + 2GIVH), where IVV and IVH are the vertically and horizontallyolarized components of probe emission at 550 nm with excitation by verticallyolarized light at 400 nm and G is the sensitivity factor of the detection system. Thexcitation and emission slits were 5 and 10 nm, respectively. The quercetin con-entration was 5 �M and HSA or HSA–p-BQ concentrations were varied between(Bartecchi et al., 1994) and 50 �M (Sengupta and Sengupta, 2002). The data weretted according to the following equation (Heyduk and Lee, 1990):

= AL + (APL − AL) X

[(LT + PT + Kd) − {(LT + PT + Kd)2 − 4LTPT)}1/2

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here A is the measured value of anisotropy; AL and APL are specific valuesf anisotropy associated with free quercetin and HSA–quercetin or HSA–p-Q–quercetin complex, respectively. The Kd value was obtained by fitting thexperimental data to the equation by using least-squares method using KYPLOTKoichi Yoshioka, 1997–2000, version 2.0, beta 13) with AL, APL and Kd as floatingarameters.

.20.2. Fluorescence quenching methodThe values of the dissociation constant (Kd) for the HSA–ligand complex (PL)

as determined by assuming a simple bimolecular binding equilibrium betweenhe HSA (P) and ligand, L, using the nonlinear least-squares method according to theollowing equation (Takita et al., 1996).

(2)�F(%) = �Fmax[L]0(Kd+[L]0) where [L]0 is the total concentration of the ligand,

L]0 = [L] + [P × L], and �F is the fluorescence intensity change observed at 340 nmhen a certain amount of the ligand is added. This is expressed as a percentage of

he fluorescence intensity of the HSA; namely �F(%) = 100 × (FPL − F0)/F0, where FPL

nd F0 are the fluorescence intensities at 340 nm of the HSA–ligand complex and theSA, respectively. The data were fitted using KYPLOT (Koichi Yoshioka, 1997–2000,ersion 2.0, beta 13) with �Fmax and Kd as floating parameters.

. Results

.1. Identification of HSA–p-BQ adduct in smokers’ serum

As reported earlier, p-BQ is derived from p-BSQ of CS by dis-roportionation (Sullivan and Reynolds, 1996) and oxidation byransition metal catalyzed proteins, including Cu, Zn-SOD andytochrome c (Banerjee et al., 2008). Conceivably, p-BQ is expectedo be produced from p-BSQ in smokers’ lung, to enter into the bloodtream and form adduct with HSA. This is evidenced by immunoblotnalysis indicating that lungs as well as serum of guinea pigsxposed to CS contain p-BQ–protein adducts (Fig. 1A and B). Welso show that serum from human smokers’ contains p-BQ adductith protein corresponding to the molecular weight of HSA (Fig. 2).

he presence of HSA–p-BQ adduct in smokers’ serum has been con-

rmed by mass spectroscopy. MALDI-TOF–TOF-MS data obtained

rom two smokers (smokers 5 and 6 of Fig. 2) show that altogetheright locations of HSA have formed adduct with p-BQ (Table 1).n separate experiments we show that HSA treated with aqueous

protein was loaded in each well. (II) ponceau S staining of the membrane as theloading control. Details of methodology are given under Section 2.

extract of CS (AECS) produces immunoblot similar to that obtainedby treatment of HSA with p-BQ (Fig. 3).

3.2. MALDI-MS analysis of HSA–p-BQ adducts

To confirm that p-BQ forms adducts with HSA, we carried outMALDI-MS of HSA incubated in vitro with different concentrationsof p-BQ. Upon complex formation with p-BQ, the molecular weightof HSA is expected to increase according to the number of p-BQadducted. For each p-BQ adduct, an increase in molecular weight(MW) of 108 (m/z) Da should be obtained. From the MALDI massspectrum of protein and p-BQ complexed protein, the increase inMW is clearly evidenced in accordance with the concentration ofp-BQ (Table 2). The number of p-BQ complexed with HSA increased

with increased molar ratio of HSA:p-BQ (Table 2). For a 1:1 molarratio of HSA:p-BQ, the number of p-BQ adducted was 1; for 1:8ratio, the number was 7; for 1:30, it was 24 and that for 1:60 it was

82 A. Ghosh et al. / Toxicology 292 (2012) 78– 89

Table 1MALDI-TOF–TOF-MS analysis of HSA–p-BQ adduct in smokers’ serum.

Amino acid residues MW of peptide MW of peptide-p–BQadduct

Number of p-BQmolecule adducted

Peptide sequence

From Upto 1

1 52 73 2497.10 2605.10 1 TCVADESAENCDKSLHTLFGDK2 94 106 1714.80 1822.80 1 QEPERNECFLQHK3 206 212 854.45 962.45 1 FGERAFK4 258 274 1941.92 2049.92 1 ADLAKYICENQDSISSK5 360 372 1552.60 1660.60 1 CCAAADPHECYAK6 390 402 1657.75 1765.75 1 QNCELFEQLGEYK7 445 466 2674.31 2782.31 1 RMPCAEDYLSVVLNQLCVLHEK8 501 521 2545.17 2653.17 1 EFNAETFTFHADICTLSEKER

Results are shown for smoker No. 5 of Fig. 1. Identical results were obtained for smoker No. 6. The detailed procedure is given in Section 2.

Table 2MALDI-MS analysis of HSA–p-BQ adducts.

Observed MW (Da) Difference (Da) Number of p-BQ in the adduct

HSA 66,539a – –HSA:p-BQ (1:1) 66,642 103 1HSA:p-BQ (1:8) 67,345 806 7HSA:p-BQ (1:30) 69,131 2593 24

4285 40

atios of p-BQ in the HSA–p-BQ complex.

4F

3N

wMait2awo(Cmraptawtb�w�Ww5tHcw

3

a

HSA:p-BQ (1:60) 70,823

a Indicates mass of intact protein before and after reaction with different molar r

0 (Table 2). The MALDI-MS profiles are given in Supplementaryig. 1.

.3. Spectrophotometry of p-BQ-adducts of amino acids and-acetyl amino acids

Previous results had indicated that p-BQ forms covalent adductsith Lys-rich regions of cytochrome c (Fisher et al., 2007). From theALDI analysis it had been observed that a large number of amino

cids in the HSA molecule were covalently modified by p-BQ. Its already known that amino acids react with quinone primarilyhrough their amino groups (Kalyanaraman et al., 1987; Bittner,006). This reactivity has even been explored to quantify proteinlso (Lichtig et al., 2001). In order to ascertain whether p-BQ reactedith �-amino groups of side chain Lys residues of HSA, we carried

ut reaction of p-BQ with the 20 common biological amino acidsSupplementary Fig. 2) and N-acetyl derivatives of Ala, Trp, Arg, His,ys and Lys (Fig. 4) and examined the chromophores spectrophoto-etrically. In all the cases except cysteine and proline, formation of

ed or brown chromophores accompanied by absorption maximat 350 nm is observed. In the case of cysteine, initially a colorlessroduct is obtained, apparently due to reductive addition of thehiol group with quinone. Proline forms a colored complex withbsorption maxima at around 540 nm. That p-BQ actually reactsith the free NH2 group is evidenced by the fact that no reac-

ion between p-BQ and amino acid occurs when the amino group islocked by acetylation. The results obtained with N-�-acetyl Ala, N--acetyl Cys, N-�-acetyl Trp, N-�-acetyl Arg and N-�-acetyl His asell as N-�-acetyl Lys and N-�-acetyl Lys are shown in Fig. 3. The N-

-amino group of Lys is equally reactive like the N-�-amino group.hen the �-NH2 group of Lys is blocked by acetylation, p-BQ reactsith the �-amino group producing absorbance at 350 nm. HSA has

9 Lys residues. Thus, the possible modification sites in HSA are par-icularly the side chain Lys residues. N-�-acetyl Arg and N-acetylis showed no characteristic peak at 350 nm, indicating that sidehain Arg and His of HSA were not involved in adduct formationith p-BQ.

.4. Estimation of free amino groups by fluorescamine

HSA has 59 Lys residues. This means that besides the N-terminusmino group, 59 free �-amino groups of Lys are potentially

Fig. 4. Absorbance spectra of N-acetyl amino acids compared with free amino acidsafter treatment with p-BQ. Five hundred nano moles of amino acids were incubatedwith 100 nmol of p-BQ in 200 �l of 20 mM potassium phosphate buffer, pH 7.4 for2 h at 37 ◦C. After incubation, 800 �l of the buffer were added to the reaction mixtureand spectra taken at 200–700 nm.

A. Ghosh et al. / Toxicology

Fam

ataiFtrewunflToo

3

ctncvop

F(

ig. 5. Changes in the HSA bound fluorescamine fluorescence intensity before andfter treatment with p-BQ; 1:1 to 1:60 indicate molar ratios of p-BQ in the HSA–p-BQixture. Details are given under Section 2.

vailable for complexation with p-BQ. With a gradual increase inhe concentration of p-BQ for complexation with HSA, there will be

gradual loss of the free �-amino groups of Lys residues. We exam-ned the loss of free amino groups by reaction with fluorescamine.luorescamine reacts with the primary amino groups of proteinso produce a fluorescent product, which gives fluorescence in theange of 400–600 with an emission maximum at 474 nm whenxcited at 390 nm (Colombo et al., 2010). It has been observed thatith gradual increase in the concentration of p-BQ, there is a grad-al loss of fluorescamine fluorescence intensity compared to theative protein (Fig. 5). About 78% decrease in the fluorescamineuorescence was observed for the HSA–p-BQ molar ratio of 1:60.hus a major reduction of available �-amino groups of Lys residuesf HSA occurred after interaction with p-BQ, indicating alkylationf Lys residues of HSA by p-BQ.

.5. Fluorescence and absorption spectra of HSA–p-BQ complex

Interaction of p-BQ with HSA produces a reddish brownhromophore having absorption maxima (�max) at 350 nm in aime-dependent manner (Fig. 6A). Trp fluorescence of HSA is sig-ificantly quenched when HSA is preincubated with increased

oncentrations of p-BQ. Fig. 6B shows that the increase in the A350alue is accompanied by concomitant decrease in the emission flu-rescence (340 nm) of Trp. The fluorescence measurements wereerformed after dilution of the solutions using shorter pathlength

ig. 6. Absorption and fluorescence spectra of HSA–p-BQ complex; (A) absorbance spectB) fluorescence intensity of different molar ratios of HSA–p-BQ complex. Details are give

292 (2012) 78– 89 83

cuvette to avoid inner filter effect (see Section 2). The intensity ofA350 value increased with increasing molar ratio of HSA:p-BQ from1:1 up to 1:60 with a concomitant decrease in Trp fluorescence ofabout 88% (Fig. 6B).There was no observable emission of p-BQ aloneunder similar conditions. The Trp emission maxima of HSA in theHSA–p-BQ complex remained unaltered showing that the decreasein fluorescence intensity was mainly due to FRET. The apprecia-ble quenching even in 1:1 molar ratio of the HSA–p-BQ complexwould indicate modification of the Lys-212 residue in the vicin-ity of Trp-214. However, an equally plausible model would be thatmodification of other Lys affects structure of the protein to quenchfluorescence.

3.6. Fluorescence and absorption spectra of peptide–p-BQcomplex

HSA has only one Trp residue (Trp-214) and Lys-212 is present inclose proximity of the Trp. To understand the effect of p-BQ on themodification of Lys-212, custom made decapeptides (see Section2) that represent the 207–216 region of the HSA molecule contain-ing the only Trp-214 residue were used. In the designed peptide,N-�-acetyl modification was made in Gly to block the N-terminal

NH2 group of Gly and thereby allowing the p-BQ molecule to reactwith the available �-amino group of the Lys residue. When onenmole of peptide #1 (CH3-CO-HN-Gly-Glu-Arg-Ala-Phe-Lys-Ala-Trp-Ala-Val-COOH) was incubated with 5 nmol of p-BQ, there wasa marked increase of A350 in the UV spectrum with a concomitant70% quenching of Trp fluorescence (Fig. 7). In contrast to this, whenLys was replaced by Gly (peptide #2, CH3-CO-HN-Gly-Glu-Arg-Ala-Phe-Gly-Ala-Trp-Ala-Val-COOH), there was neither any increase inA350 nor any quenching of Trp fluorescence (Fig. 6).

3.7. MALDI-TOF–TOF analysis of the HSA–p-BQ adduct in themolar ratio of 1:1

In order to locate the Lys residue that is most susceptible for theinitial formation of the p-BQ adduct, we employed MALDI-TOF–TOFmethod and analyzed the mass spectra obtained from the trypticdigest product of HSA:p-BQ in the molar ratio of 1:1. The MWsof the peptides expected from trypsin digestion of HSA with onemissed cleavage was obtained from GPS software and MASCOT orSWISS-PROT and 108 Da was added to the MWs to obtain the MW

of the peptide-p-BQ adduct. Analysis of the MS spectra is given inTable 3. Peptides with Lys residues showing MW of peptide-p-BQadduct have been listed. As it is not clear whether adduct formationwould change the specificity of trypsin towards Lys residues, all

ra (UV/vis) at the molar ratio HSA–p-BQ complex (1:60) at different time intervals;n under Section 2.

84 A. Ghosh et al. / Toxicology 292 (2012) 78– 89

Table 3MALDI-TOF–TOF-MS analysis of HSA–p-BQ adduct in the molar ratio of 1:1.

Amino acid residues MW (+1.0073) Adduct calculated MW Peptide sequence Adduct observed MW Ion score

From Up to

146 159 1741.8868 1850.89 HPYFYAPELLFFAK 1850.72 55.10161 174 1661.7178 1770.73 YKAAFTECCQAADK 1770.73 49.80163 174 1370.5595 1479.57 AAFTECCQAADK 1479.56 50.20206 212 853.4446 962.45 FGERAFK 962.49 779.22258 274 1940.9149 2049.92 ADLAKYICENQDSIS 2049.94 62.16467 475 1001.5505 1110.56 TPVSDRVTK 1110.47 56.47

M was

tp4t

3

sutaarlwT

Fp3gD

546 557 1341.6274 1450.63

ALDI-TOF–TOF analyses of trypsin digest of equimolar complex (1:1) of HSA:p-BQ

he Lys residues present in the peptides have been considered. Theositions of Lys residues modified by p-BQ are 159, 174, 212, 262,75, 546 or 557. Thus there is competition between the Lys residueso be modified.

.8. Fluorescence lifetime

It is well documented that the lifetime of tryptophan is sen-itive to its local environment (Engelborghs, 2001). So we havesed time-resolved fluorescence lifetime measurements of tryp-ophan to identify the existence of different protein conformationss well as to understand the Trp microenvironment of HSA beforend after complex formation with p-BQ. The fluorescence decay

aw data was fitted to a three exponential function. The averageifetime of HSA was 6.55 ns (Table 4), which is in good agreement

ith previous observations (Helms et al., 1997; Otosu et al., 2010).he average lifetime (6.55 ns) decreased gradually to 1 ns for the

ig. 7. Spectrophotometric (left) and spectrofluorometric (right) analyses ofeptide-p-BQ complex. Peptide 1 containing lysine shows increase in absorbance at50 nm and associated fluorescence quenching. Peptide 2 with lysine replaced bylycine shows no significant change in either absorbance at 350 nm or fluorescence.etails are given under Section 2.

KAVMDDFAAFVEK 1450.72 194.31

done to detect lysine residues susceptible to adduct formation.

HAS–p-BQ complex with increased concentrations of p-BQ. Thisindicates substantial change in the Trp microenvironment.

3.9. CD analysis

CD spectroscopy is widely used to gain information on pro-tein’s secondary and tertiary structure in solution. The far-UV CD(200–250 nm) of HSA shows its characteristics minima around 222and 208 nm as expected for typical �-helical protein (Fig. 8A). Withincreasing molar ratio of p-BQ in the HSA–p-BQ complex, the meanresidual ellipticity differed and the percentage of �-helix increasedfrom 65.2% to 72.3% (Table 5). The CD spectrum of a protein in thenear-UV region (250–320 nm) can be sensitive to certain aspects ofthe tertiary structure. At these wavelengths the chromophores arethe aromatic amino acids and disulfide bonds. The CD signals theyproduce are sensitive to the overall tertiary structure of the protein.The near-UV CD (250–320 nm) spectra of HSA and HSA–p-BQ com-plex indicate a significant change in the tertiary environment uponcomplex formation (Fig. 8B). Thus covalent modification of HSA byp-BQ results in secondary and tertiary structural rearrangementsof the protein.

3.10. Detection of hydrophobic region accessibility by ANS(8-anilino-1-naphthalenesulfonic acid) binding

The 8-anilino-1-naphthalenesulfonic acid (ANS) is a well knownand much utilized “hydrophobic probe” for proteins (Slavik, 1982).HSA contains two hydrophobic patches located at sub-domainIIA (site 1) and sub-domain IIIA (site 2). These patches are theprincipal site for ligand binding (Togashi et al., 2010). Using com-petitive binding assay combining two fluorescence modes, threebinding sites of hydrophobic molecules have been detected in HSA(Takehara et al., 2009). Changes seen in the fluorescence inten-sity of protein bound ANS may be attributed to alterations ofaccessibility of hydrophobic regions and ligand binding capacityof HSA–p-BQ complex. Fig. 9 shows that compared to native HSA,the ANS fluorescence decreases with increased molar ratio of p-BQin the HSA–p-BQ adduct. This indicates that the solvent accessi-ble hydrophobic regions and associated ligand binding sites of HSAbecome unavailable upon complexation with p-BQ. This result sug-gests that compared to native HSA, the ligand binding ability ofHSA–p-BQ complex is altered.

3.11. Differential scanning calorimetry (DSC) studies ofHSA–p-BQ adduct

To account for change in the stability of HSA after complexformation with p-BQ, we employed DSC method that directly

measures the transition temperature of macromolecules. Highermelting temperature generally indicates greater stability. We havefound that native HSA show three transition temperatures indicat-ing that unfolding of HSA is not simple two-state transition. DSC

A. Ghosh et al. / Toxicology 292 (2012) 78– 89 85

Table 4Fluorescence lifetimes of HSA as a function of increasing concentrations of p-BQ in the HSA–p-BQ complex.

�1 (ns) ˛1 �2 (ns) ˛2 �3 (ns) ˛3 〈�〉a (ns) �2b

HSA 3.6581 39.06 0.6337 4.72 7.5678 56.21 6.55 0.9412HSA–p-BQ (1:1) 3.0666 35.94 0.45652 5.93 7.1701 58.13 6.28 0.7963HSA–p-BQ (1:2) 2.9292 37.94 0.5581 8.93 7.0352 53.13 6.04 0.8846HSA–p-BQ (1:4) 2.2074 40.82 0.3882 15.47 6.3364 43.71 5.24 0.894HSA–p-BQ (1:8) 1.7516 40.36 0.3452 32.01 5.4537 27.63 4.08 0.8876HSA–p-BQ (1:30) 0.9072 55.37 0.3913 29.7 2.1154 14.94 1.86 1.1127HSA–p-BQ (1:60) 0.0222 62.9 0.33899 25.75 1.4416 11.31 1.00 1.208

a 〈�〉 = ˛1�1 + ˛2(2 + ˛3(3.b The magnitude of �2 denotes the goodness of the fit.

Table 5Far-UV CD analysis.

HSA HSA–p-BQ (1:1) HSA–p-BQ (1:2) HSA–p-BQ (1:4) HSA–p-BQ (1:8) HSA–p-BQ (1:30) HSA–p-BQ (1:60)

65.2 ± a1.1 66.8 ± 0.5 67.6 ± 0.7 68.9 ± 1.4 70.2 ± 0.8 71.6 ± 1.7 72.3 ± 2.1

Percent of �-helix was determined from the ellipticity at 222 nm according to Chen et al. (1972) (see Section 2). All experiments were done in triplicate.a Indicates SD.

Table 6DSC Analysis of HSA with increasing concentrations of p-BQ in the HSA–p-BQ complex.

HSA HSA–p-BQ (1:1) HSA–p-BQ (1:2) HSA–p-BQ (1:4) HSA–p-BQ (1:8) HSA–p-BQ (1:30) HSA–p-BQ (1:60)

54 58.3 60 59.8 80.2 – –

Data analyses were performed with Cpcalc software (Calorimetry Sciences Corp, Utah U.S.A.) and the temperature of the first transition is given in the table. The proteinconcentration used was ∼220 �M. For 1:30 and 1:60 molar ratios, an exothermic peak was obtained beyond 100 ◦C and thus excluded.

Fig. 8. (A) Far UV-CD and (B) near UV-CD of HSA–p-BQ complex showing change in the se2.

Fig. 9. Fluorescence of ANS bound to HSA and HSA–p-BQ complex at different molarratios (1:1 and 1:60). Details are given under Section 2.

condary and tertiary structure of HSA, respectively. Details are given under Section

measurement of HSA–p-BQ complex shows increased melting tem-perature compared to pure HSA (Table 6). Table 6 summarizes themelting temperatures (Tm) of the first transition (peak 1) obtainedfor HSA and HSA–p-BQ adducts. A consistent increase in Tm revealsan altered structure of HSA upon complexation with p-BQ. At highermolar ratios of p-BQ in the HSA–p-BQ complex (1:30 and 1:60), theprotein forms visible aggregates probably due to formation of amy-loid like structure that precipitates out of the solution accumulatingwithin the sample port. This observation indirectly supports our CDstudies. The marked increase in the �-helix content as a functionof p-BQ concentration probably makes the HSA more resistant tomelting and prone to aggregation.

3.12. Ligand binding studies

Ligand binding studies was undertaken to show whether struc-tural changes in HSA due to formation of adduct with p-BQ affectsthe binding capacity of HSA. Three different ligands were used

86 A. Ghosh et al. / Toxicology 292 (2012) 78– 89

Fig. 10. (A and C) Interaction of myristic acid and paracetamol with HSA and HSA:p-BQ complex at different molar ratios. HSA (0.5 �M) was incubated in 100 mM Tris–HClbuffer (pH 7.4) with increasing concentrations of myristic acid and paracetamol at 25 ◦C. The decrease or increase in the fluorescence intensity in percent �F% are determinedand plotted as function of ligand concentration. The excitation and emission wavelength were 295 and 340 nm, respectively. The bandpasses were 5 nm for both excitationand emission. Three experiments were conducted under the same conditions, and the plots were constructed using average values and standard deviations. Dissociationconstants were determined as described in Section 2; the solid lines are theoretical curves according to Eq. (2). (B) Steady state anisotropy was measured for quercitin (5 �M)b excitaw p-BQ co n 2; th

i(

fBHmtFdqaHpi

4ifoeefs2

efore and after addition of HSA or HSA–p-BQ complex at different molar ratios. Theavelength were 400 and 550 nm, respectively. The concentrations of HSA or HSA–

f HSA concentration. Details of the experimental procedure are given under Sectio

n this study, namely, myristic acid, quercitin and paracetamolFig. 10A–C).

Myristic acid binding shows that the dissociation constant ofatty acid gradually increases with increasing concentration of p-Q in the HSA–p-BQ adduct, indicating functional alteration ofSA due to structural perturbation. The dissociation constant foryristic acid increases from (17.57 ± 1.85) × 10−6 M for pure HSA

o (220.67 ± 15.5) × 10−6 M for HSA–p-BQ 1:8 complex (Table 7,ig. 10A). For the subsequent molar ratios (1:30 and 1:60), theissociation constant could not be determined by fluorescenceuenching due to non-detectable intensity change after ligandddition. An increase in the dissociation constant means that theSA–fatty acid complex becomes less stable due to formation of-BQ adduct. This might be a reason for the accumulation of lipid

n the blood as observed in smokers (Craig et al., 1989).Quercetin is weakly fluorescent in solution when excited at

00 nm and emission is observed at 550 nm, but the fluorescencentensity increases substantially when bound to protein. We haveound that even at 5 �M concentration, quercetin has substantialptical density at 295 and 340 nm that may cause inner filterffect during titration by quenching method. Therefore we have

mployed the fluorescence anisotropy method which is suitableor measuring protein–ligand dissociation constant and much lessensitive to inner filter effects than intensity (Jameson and Mocz,005). The anisotropy value increased as a function of protein

tion and emission slits were 5 and 10 nm, respectively. The excitation and emissionomplex were varied from 1 to 50 �M. Anisotropy values were plotted as a functione dissociation constants were determined according to Eq. (1).

concentration and reached a plateau indicating saturation of bind-ing sites (Fig. 10B). We observed that initially at low concentrationsthe dissociation constants decreased (Kd) indicating stabilizationof the complex but the values increased substantially and contin-uously beyond HSA:p-BQ molar ratio of 1:4 showing less affinityof HSA for quercetin (Table 7). These results indicate that in heavysmokers, where the molar ratio of HSA:p-BQ is expected to be high,the concentration of free quercitin in the blood would be high.

In the case of paracetamol binding, a greater stability ofprotein–drug complex (lower Kd) was observed for lower molarratios of p-BQ (up to 1:8) (Table 7). This stability may have adverseeffects as the drug release will be low causing less availability of thefree drug concentration in the blood. On the other hand, for highermolar ratios of p-BQ (1:30 and 1:60) in the HSA–p-BQ complex, thedissociation constant decreased markedly (Table 7, Fig. 10C), indi-cating that under this condition the concentration of the drug inthe blood may reach a very high level.

4. Discussion

In this paper we show that in smoker’s blood, p-BQ forms cova-lent adducts with HSA. p-BQ is not present in cigarette smoke (CS).It is apparently produced from p-BSQ, a long-lived semiquinoneradical present in substantial amounts in cigarette smoke (Pryor

A. Ghosh et al. / Toxicology 292 (2012) 78– 89 87

Table 7Dissociation constant (×10−6) in molar (M) as determined by fluorescence study.

HSA HSA–p-BQ (1:1) HSA–p-BQ (1:2) HSA–p-BQ (1:4) HSA–p-BQ (1:8) HSA–p-BQ (1:30) HSA–p-BQ (1:60)

Myristic acid 17.6 ± b1.8 50.6 ± 2.5 83.3 ± 4.3 112.7 ± 7.0 220.7 ± 15.5 –a –a

Quercetin 13.8 ± 2.9 16.0 ± 4.51 16.4 ± 4.7 18.9 ± 2.2 22.8 ± 6.2 53.0 ± 17.6 58.7 ± 20.3Paracetamol 119.2 ± 4.6 76.8 ± 9.3 47.1 ± 2.7 44.2 ± 5.6 23.9 ± 2.5 2239.3 ± 330.4 4677.7 ± 474.18

T one a

eTgasmsiiLi

pwa1atohLcm

bbaBbnLartaid

tpddtppDswbotca

fb

he HSA concentration was 0.5 �M in all cases. At least three measurements were da Indicates no fluorescence change could be detected.b Indicates SD.

t al., 1983; Chatterjee, 2005; Banerjee et al., 2008; Dey et al., 2010).he results obtained with lung tissue and serum from CS-exposeduinea pigs as well as serum from human smokers indicates thatfter CS exposure BQ is produced in the lungs, enters the bloodtream and forms adduct with HSA. We further show that for-ation of HSA–p-BQ adducts results in alteration of the protein’s

econdary and tertiary structure and impairment of ligand bind-ng properties. The mechanism of formation of HSA–p-BQ adducts apparently Michael addition of p-BQ with the �-NH2 groups ofys residues. His and Arg residues of the protein are not involvedn Michael addition with p-BQ.

MALDI analysis showed that when HSA was incubated with-BQ in the molar ratio of 1:1, the p-BQ adducted preferentiallyith Lys-212, which is very near to the sole Trp-214. When HSA

nd p-BQ were incubated with increased molar ratios from 1:1 to:60, there was a gradual increase in the number of p-BQ moleculettached to HSA (up to 40), which was accompanied by concomi-ant increase in the quenching of Trp fluorescence. The quenchingf the Trp-214 fluorescence indicates that the conformation of theydrophobic binding pocket in subdomain IIA is affected (Trynda-emiesz, 2004). The decrease in Trp lifetime of HSA in the HSA–p-BQomplex further reinforces our conclusion regarding change in theicro environment around the Trp residue.Covalent modification of Lys residues by p-BQ is accompanied

y perturbation of the secondary structure of HSA as demonstratedy the far-UV CD spectra. Also the near-UV spectra indicate thatfter complexation with p-BQ, the tertiary structure is changed.oth these changes lead to a decrease in the number of hydropho-ic pockets as observed from ANS binding studies. This result isot unexpected considering the fact that covalent modification ofys residues by p-BQ alters the overall charge of the HSA moleculend many of the electrostatic interactions are expected to be lostesulting in changes in the secondary and tertiary conformation ofhe protein. Again p-BQ modification will cause a huge steric effects shown in a model of cytochrome c–p-BQ complex, changing thentermolecular distance between the residues and causing furtheramage in the structure (Fisher et al., 2007).

DSC studies of HSA showed three distinct peaks indicating thathe unfolding process of this protein is not a simple two-staterocess. The peaks might be indication of sequential melting ofifferent domains or may be indicative of the presence of interme-iates. DSC data show that upon complex formation with p-BQ, allhe three transition temperatures of HSA significantly increase. Arobable explanation is alteration of the structure of HSA after com-lexation with p-BQ, lending further support to our CD data. TheSC experiment also indicated that the ensembles at latter tran-

ition temperatures were more populated compared to free HSA,here the first transition ensemble was predominant. Thus pertur-

ation of structure of HSA by p-BQ not only affected the native statef HSA but also influenced either the domain-domain interaction orhe intermediate state. It might be concluded that covalent modifi-ation of HSA by p-BQ led to a global effect rather than simply local

lterations.

Interaction with p-BQ not only alters the structure and con-ormation of HSA, but also its ligand binding capacity. This haseen revealed by experiments with myristic acid (MA) as a

nd standard deviations were calculated.

representative fatty acid. The protein conformation of HSA–MAcomplex is very similar to that obtained by interaction of HSA witha variety of other fatty acids, including palmitic acid and oleic acid(Bhattacharya et al., 2000). So, the HSA–MA complex would displaythe effect of the conformational changes of HSA–fatty acid bindingin general. We have indicated that the initial p-BQ molecule formscovalent adduct predominantly with Lys 212. It is also known thatTrp 214 is involved in binding of MA molecule (Bojko et al., 2008).We found that the apparent dissociation constant of MA bindingincreased with increasing molar ratio of p-BQ in the HSA–p-BQcomplex. This would indicate that binding of fatty acid with theHSA–p-BQ adduct is less stable. This might be one reason for accu-mulation of lipid in the smoker’s blood (Craig et al., 1989) leadingto atherosclerosis.

The impairment of binding of other two ligands examined,namely quercetin and paracetamol, implied that p-BQ mediatedcovalent modification in HSA may affect the binding, transport andbioavailability of these two ligands in the smokers.

Quercitin (3,5,7,3′,4′-pentahydroxyflavone) is a member of nat-urally occurring widely distributed compounds, the flavonoids,which are found in plants, fruits, flowers and plant derived foods(Harborne and Williams, 2000). Abundant in the human diet,quercitin possesses various biological and biochemical propertiesincluding antioxidant, anti-inflammatory, antineoplastic and car-dioprotective activities (Cook and Samman, 1996; Middleton et al.,2000; Dangles et al., 2001; Yang et al., 2001). As the most abundantcarrier protein, HSA plays an important role in binding, transportand disposition of quercitin (Manach et al., 1995; Kaldas et al.,2005). However, when the structure of HSA is modified, as occursin the smokers’ blood due to formation of HSA–p-BQ adduct, thebinding and distribution of quercitin is expected to be affected.The dissociation constant of HSA–p-BQ–quercitin complex initiallydecreased at low molar ratio of p-BQ, but increased fairly highat higher p-BQ concentration. Low dissociation constant indicatesgreater stability of the HSA–p-BQ–quercitin complex indicatingthat the level of quercitin in the blood and thereby its availabil-ity would be less. On the contrary, in heavy smokers the molarratios of p-BQ in the HSA–p-BQ adduct would be expected to behigh resulting in less binding of quercitin. Under this condition,the concentration of free quercitin in the blood would be highthat might eventually cause toxicity. Quercetin is an abundantflavonoid in the human diet with numerous biological activities,which may contribute to the prevention of human disease butalso may be potentially harmful. In the presence of hydrogen per-oxide/peroxidases, quercetin is oxidized and covalently links toproteins with particular high affinity for HSA and thereby acts as acytotoxic prooxidant (Metodiewa et al., 1999; Kaldas et al., 2005).

Paracetamol (N-acetyl-p-aminophenol) is a commonly usedanalgesic and antipyretic drug. Paracetamol has been in use as ananalgesic for home medication for over 30 years and is acceptedas a very effective treatment for the relief of pain and fever inadults and children. It is the most used medicine after acetylsalicylic

acid in many countries as an alternative to aspirin and phenacetin(Kragh-Hansen et al., 2002; Daneshgar et al., 2009). It is knownthat binding to HSA controls the free, active concentration of adrug, and provides a reservoir for a long duration of action, and

8 cology

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ltimately affects drug absorption, metabolism, distribution, andxcretion. The binding property of paracetamol showed some pecu-iarities. As observed with quercitin, the dissociation constant ofSA–p-BQ–paracetamol complex initially decreased at low molar

atio of p-BQ, but increased fairly high at higher p-BQ concentra-ion. Though lower dissociation constant indicates greater stabilityf the HSA–paracetamol complex, but it also has physiologicallydverse effect leading to less release of the amount free drug in thelood of smokers. In a light or mild smoker, the concentration ofSA–p-BQ is expected to be comparatively low. Thus a light or mild

moker may have to consume more paracetamol to get the simi-ar clinical effect compared to a non-smoker. In heavy smokers, aigh molar ratio of p-BQ in the HSA–p-BQ–paracetamol adduct isxpected to occur. Since the dissociation constant at high molaratio of p-BQ is markedly increased, the binding and transport ofhe drug will be less resulting in high concentration of free parac-tamol in the blood of heavy smokers. This might eventually causeepatotoxicity of paracetamol in heavy smokers. It is known thataracetamol produces necrosis of the centrilobular cells of the liverhen taken in overdose (Cook and Samman, 1996; Bessems andermeulen, 2001; James et al., 2003).

. Conclusion

Results obtained with CS-exposed guinea pigs as well as humanmokers indicate that after CS exposure p-BQ is produced in theungs, enters the blood stream, and forms covalent adduct witherum albumin. p-BQ is not present in CS. It is apparently formedrom p-BSQ of CS after oxidation by transition metal containingroteins in the lungs. p-BQ forms covalent adducts with �-aminoroups of the lysine residues of HSA in a concentration dependedanner. This results in alterations of the secondary and tertiary

tructures of HSA leading to impairment of binding with ligands,ncluding myristic acid, quercitin and paracetamol. The impairmentf binding may affect the transport, distribution and metabolism ofhe ligands.

onflicts of interest

The authors declare that they do not have any conflicts of inter-st.

cknowledgments

We gratefully acknowledge David M. Jameson (University ofawaii at Manoa) for critically reading the manuscript before

ubmission; Prof. Somen Basak, Saha Institue of Nuclear Physics,olkata, for CD analysis; Dr. R Nagaraj, CCMB for providing someALDI-MS data; GlaxoSmithKline Pharmaceuticals Limited, India,

or a gift of pure paracetamol.Funding: This research was supported by CSIR, Government of

ndia, grant no. 37(1368)/09-EMR-II and Juthika Research Founda-ion of Calcutta University. AG (PhD scholar) is a senior researchellow of CSIR; AC (PhD scholar; AD (PhD scholar) is Juthikaesearch Fellow; ND (PhD scholar) a CSIR senior research fellow;

BC is INSA Honorary Scientist. The funders had no role in studyesign, data collection and analysis, decision to publish or prepa-ation of the manuscript.

Contributors: IBC conceived and designed major part of thexperiments. RB designed all the experiments of biophysical stud-

es. AG performed most of the experiments. AC did a part of theiophysical studies. RC, TD, KP and AD did the proteomics studies.SC did the DSC experiments. IBC and RB analyzed the data. IBContributed reagents/materials/analysis tools and wrote the paper.

292 (2012) 78– 89

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.tox.2011.11.014.

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