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International Journal of Biological Macromolecules 59 (2013) 349– 356

Contents lists available at SciVerse ScienceDirect

International Journal of Biological Macromolecules

jo ur nal homep age: www.elsev ier .com/ locate / i jb iomac

eroxynitrite induced structural changes result in the generation ofeo-epitopes on human serum albumin

arvez Ahmad, Moinuddin, Asif Ali ∗

epartment of Biochemistry, Faculty of Medicine, Aligarh Muslim University, Aligarh, U.P., India

a r t i c l e i n f o

rticle history:eceived 2 March 2013eceived in revised form 22 April 2013ccepted 23 April 2013vailable online 1 May 2013

a b s t r a c t

Human serum albumin (HSA), the most abundant plasma protein, is quite vulnerable to oxidizing andnitrating agents. In this study, peroxynitrite induced nitration and oxidation of HSA was assessed byvarious physicochemical techniques. Cross-linking of HSA was evident on polyacrylamide gel elec-trophoresis. The carbonyl content was markedly elevated in peroxynitrite-modified HSA as comparedto the native protein. Dityrosine and 3-nitrotyrosine were present only in peroxynitrite-modified HSA.

eywords:uman serum albumineroxynitriteitrotyrosinearbonyl

The peroxynitrite-modified HSA induced high titre antibodies in experimental animals showing highspecificity towards the immunogen. Spectroscopic studies showed structural alterations in the HSAmolecule upon peroxynitrite treatment which result in the generation of neo-epitopes and enhancedimmunogenicity. The possible role of damaged HSA in various diseases has been suggested.

© 2013 Elsevier B.V. All rights reserved.

pectroscopy

. Introduction

A major and aggressive reaction of nitric oxide (NO•) is withuperoxide anion (O2

•−) to form peroxynitrite (ONOO−) which has physiological half-life of approximately 1 s, as it decomposespontaneously to give nitrate [1].

2•− + NO• → ONOO−

The possible biological significance of this reaction was firstealized by Beckman and co-workers who pointed out that per-xynitrite may be formed under pathophysiological conditions,here both NO• and O2

•− are produced at high rates by phagocyticells such as macrophages, and that ONOO− is a potent oxidantith the potential to destroy critical cellular components [2]. Per-

xynitrite can alter a variety of biomolecules but possesses highffinity for tyrosine residues in proteins, and 3-nitrotyrosine is

relatively specific marker of peroxynitrite-mediated damage toroteins [3]. Reaction of peroxynitrite with tyrosine residues is aovalent modification that results in attachment of a nitro ( NO2)roup on the aromatic ring of tyrosine residues [4]. Nitrotyrosine

ormation is considered as a marker of nitrosative stress. Morehan 60 human disorders are now known to be associated withrotein nitration. Indeed, the formation of protein 3-nitrotyrosine

∗ Corresponding author: Department of Biochemistry, Faculty of Medicine, J.N.edical College, Aligarh Muslim University, Aligarh 202002, U.P., India.

el.: +91 941 227 3580; fax: +91 571 272 0030.E-mail address: asifali amu@rediffmail.com (A. Ali).

141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.ijbiomac.2013.04.068

in vivo has been shown in a number of inflammatory conditionsin human and experimental animals [5]. Peroxynitrite also causesoxidation of sulfhydryls [6]. Being an oxidizing agent, peroxyni-trite also causes protein oxidation; the most prominent markerof protein oxidation is increase in the carbonyl content of pro-teins which is the outcome of oxidative modifications of the sidechains of lysine, proline, arginine and threonine [7]. Increase in pro-tein carbonyls has been observed in many diseases like diabetes,Alzheimer’s, systemic lupus erythematosus, rheumatoid arthritis,sepsis, chronic renal failure, cancer, etc. [8]. Local pH and microen-vironment affect the reactions of peroxynitrite, with hydrophobicmembrane compartments favouring nitration and aqueous envi-ronments favouring oxidation.

Human serum albumin (HSA) is the most abundant proteinfound in human plasma. It is a heart-shaped single polypeptide of66 kDa. It has 585 amino acid residues with eighteen tyrosines, sixmethionines, one tryptophan, seventeen disulfide bridges and onlyone free cysteine (Cys34). HSA carries out several clearly definedphysiological functions like maintenance of colloidal osmotic pres-sure, free radical scavenging, binding and transport of importantsolutes, etc. [9]. Several studies have shown that HSA is quitevulnerable to modification by reactive oxygen species and an ele-vated level of oxidized albumin is found in various diseases [10]. Ithas been shown that peroxynitrite preferentially reacts with thecysteine residue of HSA and causes thiol oxidation [11]. HSA is

continuously exposed to oxidative stress conditions due to its abun-dance in the plasma, leading to the conformational and functionalalterations of the protein molecule. The altered HSA may con-tribute to the progression of many diseases. In the present study,

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e have investigated the quantitative and qualitative changes inhe structure of HSA upon reaction with peroxynitrite causing theeneration of neo-epitopes in protein which in turn induce theroduction of highly specific antibodies in experimental animals.

. Materials and methods

.1. Chemicals

Human serum albumin, 3-nitrotyrosine, sodium nitrite, 1-nilinonaphthalene-8-sulphonic (ANS) acid, tyrosine, tryptophan,henylalanine, protein A-agarose affinity column, p-nitrophenylhosphate, Tween-20, Freund’s complete and incomplete adju-ants, and 2,4-dinitrophenyl hydrazine (DNPH) were obtainedrom Sigma–Aldrich (St. Louis, MO). Hydrogen peroxide, sodiumydroxide, silver nitrate and guanidine hydrochloride were fromualigens (Mumbai, India). Flat bottom polysorp ELISA modulesere purchased from NUNC, Denmark. All other reagents were of

he highest analytical grade available.

.2. Peroxynitrite-modification of HSA

Peroxynitrite was synthesized by rapid quenched flow processsing sodium nitrite and acidified hydrogen peroxide [12] andtored in 1.2 M NaOH at −20 ◦C. Before each use, concentration oftored peroxynitrite was determined from absorbance at 302 nmsing molar extinction coefficient of 1670 M−1 cm−1. HSA, at aoncentration of 5 �M in phosphate buffered saline (PBS) (10 mModium phosphate buffer, 150 mM NaCl, pH 7.4), was incubatedith 60, 125, 250, 500 and 750 �M peroxynitrite at 37 ◦C for 3 h.

he reaction volume was made up to 3 ml with PBS. After incuba-ion, the solutions were extensively dialyzed against PBS to removexcess peroxynitrite. To maintain the same condition for the con-rol, unmodified HSA was also dialyzed in the same manner. TheSA concentration in both the cases was determined spectrophoto-etrically using a molar extinction coefficient of 35,219 M−1 cm−1

t 280 nm [13].

.3. Absorbance spectroscopy

The absorption profiles of native and peroxynitrite-modifiedamples were recorded on Shimadzu UV-1700 spectrophotometern the 200–400 nm wavelength range.

.4. Electrophoresis

Native and peroxynitrite-modified HSA were analyzed byodium dodecyl sulphate polyacrylamide gel electrophoresisSDS–PAGE) under non-reducing conditions on a 10% polyac-ylamide gel, as described previously [14]. Electrophoresis waserformed at 80 V for 4 h at room temperature and the proteinands in the gel were visualized by silver staining.

.5. Fluorescence studies

Fluorescence spectra were recorded on Hitachi F-200 spec-rofluorimeter at 25 ± 0.1 ◦C. Fluorescence of tyrosine residues inative and peroxynitrite-modified HSA was monitored after exci-ation at 275 nm and recording the emission in the 300–400 nmange. Loss in fluorescence intensity (FI) was calculated from theollowing equation:

loss of FI =(

FInative sample − FImodified sample

FInative sample

)× 100

ical Macromolecules 59 (2013) 349– 356

2.6. Effective protein hydrophobicity

Binding of ANS to native and peroxynitrite-modified HSA wasevaluated in terms of fluorescence. The ANS fluorescence is affectedby exposure or masking of hydrophobic patches in proteins. Afresh stock solution of ANS was prepared in distilled water andits concentration was determined spectrophotometrically usinga molar extinction coefficient of 5000 M−1 cm−1 at 350 nm [15].The molar ratio of protein and ANS was 1:10 and emission spec-tra were recorded in the range of 400–600 nm using an excitationwavelength of 380 nm. Decrease in fluorescence intensity (FI) wascalculated as follows:

% loss of FI =(

FInative sample − FImodified sample

FInative sample

)× 100

2.7. Estimation of dityrosine

The dityrosine content of peroxynitrite-modified HSA wasdetermined spectrophotometrically using a molar extinction coef-ficient of 4000 M−1 cm−1 at 330 nm [16]. Dityrosine formation wasalso confirmed by fluorescence spectra after excitation of nativeand peroxynitrite-modified HSA at 330 nm and measuring emissionin the 350–450 nm range.

2.8. Nitrotyrosine determination

The concentration of nitrotyrosine in peroxynitrite-modifiedHSA was determined by measuring the absorbance at 420 nm usinga molar extinction coefficient of 4300 M−1 cm−1 [17].

2.9. Determination of protein-bound carbonyl groups

Carbonyl content of native and peroxynitrite-modified HSA wasdetermined after reaction with DNPH [18]. The final absorbancewas read at 360 nm against appropriate blank. The carbonylcontent was determined using a molar extinction coefficient of22,000 M−1 cm−1 and expressed as nmol/mg protein.

2.10. Circular dichroism

CD measurements were carried out on a Jasco spectropo-larimeter (J-815) equipped with a Jasco Peltier-type temperaturecontroller (PTC-424S/15). The instrument was calibrated with d-10-camphorsulphonic acid. Spectra were taken in a cell of 1 and10 mm path length and protein concentrations used were 4.5 and12 �M for far and near-UV CD, respectively. The results wereexpressed as mean residue ellipticity (MRE) in deg cm2 dmol−1

which is defined as:

MRE = �obs(m deg)10 × n × C × l

where �obs is the CD in milli-degree, n is the number of amino acidresidues (585 − 1 = 584), l is the path length of the cell, and C is theconcentration of protein in moles/litre. Helical content was calcu-lated from the MRE values at 222 nm using the following equation[19]:

% ˛-helix =(

MRE222 nm − 234030, 300

)× 100

2.11. FTIR spectroscopy

Native and peroxynitrite-modified HSA samples were firstlyophilized and prepared as KBr pellets and then subjected to spec-tral recording on a Tensor 37 FTIR spectrometer (Bruker).

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.12. Immunization schedule

White New Zealand female rabbits were immunized withative and peroxynitrite-modified HSA as described previously20]. Briefly, rabbits (n = 4; two each for native and peroxynitrite-

odified HSA) were immunized intramuscularly at multiple sitesith 100 �g of antigen emulsified with an equal volume of Fre-nd’s complete adjuvant. The booster doses were administered inreund’s incomplete adjuvant at weekly intervals for 6 weeks withhe same amount of antigen. Test bleeds, performed 7 days postoost, gave significant antibody titre. The sera were heated at 56 ◦Cor 30 min to inactivate complement proteins and stored at −20 ◦Cith 0.1% sodium azide as preservative. This study had clearance

rom bioethical committee of the institution.

.13. Protein A-agarose affinity chromatography

IgG from rabbit sera was isolated by affinity chromatography onrotein A-agarose affinity column [21]. Protein-A (from Staphylo-occus aureus) binds to IgG from most mammalian species throughnique histidine residues present in Fc region of the molecule.he column was pre-equilibrated with PBS and 0.3 ml serum waspplied on it. The wash through was recycled 2–3 times andnbound material was removed by extensive washing with PBS.he bound IgG was eluted with 0.58% (v/v) acetic acid in 0.85%w/v) sodium chloride and collected in tubes containing 1.0 ml of.0 M Tris–HCl, pH 8.5. Three ml fractions were collected and theirbsorbance was read at 278 nm. The IgG concentration was deter-ined considering 1.4 OD278 = 1.0 mg IgG/ml. The homogeneity of

solated IgG was checked on 10% SDS–PAGE. The isolated IgG wasialyzed against PBS and stored at −20 ◦C with 0.1% sodium azide.

.14. Enzyme linked immunosorbent assay

ELISA was performed on flat bottom polystyrene plates asescribed earlier [22]. Polystyrene PolySorp immunoplates with 96ells were coated with 100 �l of native or peroxynitrite-modifiedSA (10 �g/ml) in antigen coating buffer. The plates were incubated

or 2 h at 37 ◦C and overnight at 4 ◦C. Each sample was assayed inuplicate and half of the plate, devoid of antigen coating, served asontrol. The wells of the test plates were washed three times withBS-T (20 mM Tris, 2.68 mM KCl, 150 mM NaCl, pH 7.4, containing.05% Tween-20) and unoccupied sites were blocked with 150 �lf 1.5–2.5% fat-free skimmed milk in TBS (10 mM Tris, 150 mMaCl, pH 7.4) for 4–6 h at 37 ◦C. After incubation, the plates wereashed 5–6 times with TBS-T. Test serum, serially diluted in TBS-

, was added to each well (100 �l/well) and reincubated for 2 h at7 ◦C and then overnight at 4 ◦C. After incubation, the plates wereashed three times with TBS-T and bound antibodies were assayedith anti-rabbit alkaline phosphatase conjugate in TBS. After 2 h

ncubation, the plates were again washed three times with TBS-Tnd twice with distilled water and then coated with p-nitrophenylhosphate (substrate). Absorbance (A) in each well was monitoredt 405 nm on an automatic microplate reader. Each sample was runn duplicate. Results were expressed as mean of Atest − Acontrol.

.15. Competition ELISA

The antigenic specificity of antibodies was determined by com-etition ELISA [23]. The plates were coated with 100 �l of antigennative or peroxynitrite-modified HSA, 10 �g/ml) for 2 h at 37 ◦Cnd overnight at 4 ◦C. Varying amounts of inhibitors (0–20 �g/ml)

ere mixed with a constant amount of antiserum or affinity puri-ed IgG. The mixtures were incubated at 37 ◦C for 2 h and overnightt 4 ◦C. The immune complex thus formed was coated in the wellsnstead of serum. The remaining steps were the same as mentioned

ical Macromolecules 59 (2013) 349– 356 351

above in direct binding ELISA. Percent inhibition was calculatedusing the equation:

Percent inhibition = 1 −(

Ainhibited

Auninhibited

)× 100

2.16. Band shift assay

Band shift assay was performed for the visual detection ofantigen–antibody interaction and immune complex formation [24].Immune complex was prepared by incubating constant amountof native or peroxynitrite-modified HSA with varying amounts ofaffinity purified anti-peroxynitrite-modified HSA IgG in PBS for2 h at 37 ◦C and overnight at 4 ◦C. The samples were then elec-trophoresed on 10% SDS–PAGE for 4 h at 80 V and the protein bandswere visualized by silver staining.

2.17. Statistical analysis

Data are presented as mean ± standard deviation (SD). Statisti-cal significance of data was determined by Student’s t test and a pvalue of <0.05 was considered as significant.

3. Results and discussion

During oxidative burst triggered by inflammation, the cells ofimmunoregulatory network produce both nitric oxide and super-oxide. These two free radicals can combine to produce peroxynitritewhich causes nitration of tyrosine residues and oxidation of pro-teins. Tyrosine nitration can profoundly alter protein functionindicating that this reaction may be fundamentally related to oxida-tive cell injury. Therefore, it is a subject of great interest to studytyrosine nitration of HSA because formation of 3-nitrotyrosinein vivo in various pathological conditions has been well established[25] and it is for this reason that 3-nitrotyrosine is considered tobe a relatively specific marker of nitrosative damage mediated byperoxynitrite. In addition to nitration, peroxynitrite is also a potentoxidizing agent and can oxidize a variety of biomolecules includ-ing proteins. Many studies have shown the potent role of reactiveoxygen species (ROS) and reactive nitrogen species (RNS) in var-ious human diseases including cancer development [26] whichcan be delayed/prevented by the use of anti-oxidants [27]. HSAis continuously exposed to oxidative stress, which can alter theconformation and function of this molecule, resulting in modifi-cation of its biological properties and hence its contribution tothe progression to different diseases. Therefore, characterization ofperoxynitrite induced structural and immunological modificationin HSA assumes significance.

3.1. Physicochemical characterization of peroxynitrite-modifiedHSA

Native HSA showed an absorbance peak at 278 nm whichexhibited hyperchromicities upon modification by different con-centrations of peroxynitrite (Fig. 1). Repeated experimentsexhibited little difference between the hyperchromicities inducedby 500 and 750 �M peroxynitrite. Therefore, HSA modified with500 �M peroxynitrite was used in further studies. Hyperchromic-ities at 278 nm could be attributed to peroxynitrite-induceddenaturation due to oxidation and nitration of native HSA. Theabsorbance profile of peroxynitrite-modified HSA also showedpeak at 420 nm, which is a characteristic of 3-nitrotyrosine (inset).

Furthermore, the generation of yellow colour in samples ofperoxynitrite-modified HSA (with absorption peak at 420 nm) alsoreflects the formation of nitrotyrosine. The nitrotyrosine contentin modified HSA was calculated to be 29.6 ± 2.3 nmol/mg protein.

352 P. Ahmad et al. / International Journal of Biolog

Fig. 1. Absorbance spectra of native HSA (-�-) and HSA modified by 60 �M (-�-),1so

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25 �M (-�-), 250 �M (-�-), 500 �M (-�-) and 750 �M (-©-) peroxynitrite. Insethows absorbance profile of standard 3-nitrotyrosine. The spectra are the averagef three determinations.

t is quite clear from the results that peroxynitrite-modificationaused concentration dependent generation of nitrotyrosine andonsequent alteration in the native structure of HSA.

Native and peroxynitrite-modified HSA was subjected to non-educing polyacrylamide gel electrophoresis under denaturingonditions (Fig. 2). Native HSA moved as compact single bandhile HSA, modified with increasing concentrations of peroxy-itrite, showed thickening of bands as compared to the nativerotein. HSA modified with 500 �M peroxynitrite (Fig. 2, lane 5)howed maximum band width. It is quite clear that peroxyni-rite induced modification (nitration and oxidation) has producedignificant structural changes in HSA. The gradual thickening ofands, as compared to the native HSA, may be due to the intra-

olecular cross-linking in the modified protein, which might lead

o abnormal migration of the protein molecules. The cross-linkingay be formed due to the interactions of one tyrosyl radicalith another tyrosyl radical, generating O,O′-dityrosine covalent

ig. 2. SDS–polyacrylamide gel electrophoresis of HSA under non-reducing con-itions. Protein samples (10 �g in each lane) were loaded on gel. Electrophoresisas performed on 10% SDS–PAGE for 4 h at 80 V. Lanes: (1) native HSA; (2–5) HSAodified with 60, 125, 250 and 500 �M peroxynitrite.

ical Macromolecules 59 (2013) 349– 356

cross-links [28]. Cross-linking and oligomerization have also beenreported during nitration of several other proteins [29].

The formation of dityrosine was confirmed from fluorescencespectra obtained when the samples were excited at 330 nm. Aprominent emission peak at 401 nm in case of peroxynitrite-modified HSA was seen which was completely absent in the nativeprotein (Fig. 3A). The dityrosine concentration of peroxynitrite-modified HSA was found to be 112.7 ± 3.1 nmol/mg protein. Thisresult, along with the SDS-PAGE analysis, confirmed the formationof O,O′-dityrosine upon peroxynitrite modification of HSA. This maybe the reaction of one tyrosyl radical generated by peroxynitritewith another tyrosyl radical of HSA molecule [16].

Peroxynitrite-modified HSA showed 79% loss in fluorescenceintensity and a blue shift of 34 nm as compared to its native formwhen excited at 275 nm (Fig. 3B). The loss could be ascribed to thedestruction of tyrosine residues and/or modification of the tyrosinemicro-environment upon peroxynitrite modification.

The conformational changes in HSA and measurement of itshydrophobic surface accessibility were studied by ANS bindingexperiment. ANS is an extrinsic probe which can bind to apolarsites on proteins. It gives high fluorescence emission upon bindingto hydrophobic regions, but does not fluoresce when present freein solution or in denatured protein solution [30]. Compared to thenative protein, peroxynitrite-modified HSA showed 88.2% loss influorescence intensity indicating that native HSA possesses a num-ber of hydrophobic patches on its surface which were disruptedupon treatment with peroxynitrite (Fig. 3C). The peroxynitriteinduced structural perturbations in HSA molecule led to the lossof ANS binding sites [31].

Carbonyl content is the most commonly used marker of pro-tein oxidation and its accumulation has been observed in manyhuman diseases [32]. The typical markers of protein oxidation arecarbonyls of lysine, arginine, threonine and proline residues whichare detected after their derivatization with 2,4-dinitrophenylhydrazine. The reaction forms a stable 2,4-dinitrophenyl hydrazonewhich is measured spectrophotometrically at 360 nm. The carbonylcontents of native and peroxynitrite-modified HSA were found tobe 3.1 ± 0.4 and 20.24 ± 1.2 nmol/mg protein, respectively (Fig. 4).This corresponds to almost seven fold increase in carbonyl levelas compared to native HSA. The result suggests that peroxynitriteinduced oxidation of HSA may be responsible for alterations in itsstructural and biological properties.

The secondary structure of HSA was monitored by far-UV CDexperiment which showed a decrease in percent helicity of HSAupon peroxynitrite modification. The CD spectra of HSA exhibitedtwo minima, at 208 and 222 nm, which is typical for �-helical struc-ture (Fig. 5A). The �-helix content was 64% in native HSA and 45%in peroxynitrite-modified HSA. This change in secondary structureshows that HSA undergoes conformational changes in the presenceof peroxynitrite.

The tertiary structure of proteins is inferred by near-UV CD spec-tra which are sensitive to the environment of aromatic side chains[33]. The native HSA showed a peak at around 275 nm, which is spe-cific for tyrosine residues. However, this peak was less pronouncedin peroxynitrite-modified HSA (Fig. 5B) which shows that tyrosineresidue(s) is/are modified by peroxynitrite. The above modificationin tyrosine residue(s) might be responsible in altering the tertiarystructure of HSA and hence, may cause alterations in its physiolog-ical functions.

The changes in the secondary structure of HSA were further con-firmed by FTIR, in which bands originating due to amide I and amideII vibrations were analyzed. The amide I band, which arises due to

C O stretching, is particularly important in the secondary structurestudies [34]. Upon peroxynitrite modification of HSA, the amideI band was shifted from 1656 to 1638 cm−1 while a shoulder ofamide II band, which arises due to CN stretching and NH bending,

P. Ahmad et al. / International Journal of Biological Macromolecules 59 (2013) 349– 356 353

Fig. 3. Fluorescence emission spectra of native (-�-) and peroxynitrite-modifiedHSA (-�-) at different excitation wavelengths; (A) at 330 nm for dityrosine, (B) at275 nm for tyrosine, and (C) at 380 nm for ANS binding to native and peroxynitrite-m

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Fig. 4. Carbonyl content of native and peroxynitrite-modified HSA. The bars showaverage carbonyl content (±SD) of three independent assays.

Fig. 5. Far-UV (A) and near-UV (B) CD spectra of native (-�-) and peroxynitrite-

odified HSA. The spectra are the average of three determinations.

hifted from 1553 to 1546 cm−1 (Fig. 6). It is clear from these resultshat amide I is more sensitive than amide II towards peroxynitrite

odification. The observed shifting in amide I and amide II bandss a clear proof of secondary structure perturbation and fully sup-orts the observations of far-UV CD spectra. The characterization

modified HSA (-�-). The spectra are the average of three determinations.

354 P. Ahmad et al. / International Journal of Biological Macromolecules 59 (2013) 349– 356

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Fig. 7. Level of induced antibodies against native and peroxynitrite-modified HSA.

ig. 6. FTIR spectra of (A) native and (B) peroxynitrite-modified HSA. The spectrare the average of three determinations.

f native and peroxynitrite-modified HSA has been summarized inable 1.

.2. Immunogenicity of native and peroxynitrite-modified HSA

The immunogenicity of peroxynitrite-modified HSA was evalu-

ted by inducing antibodies in female rabbits. The direct bindingLISA result showed antibody titre of >1:12,800 (Fig. 7B) as com-ared to <1:6400 titre obtained in the case of native HSA (Fig. 7A).

able 1haracterization of native and peroxynitrite-modified HSA.

Parameters Native HSA Peroxynitrite-modifiedHSA

Absorbance (at 278 nm) 0.164 0.235Fluorescence intensity, FIa(�ex = 275)

114.77 22.25

ANS-binding, FIa(�ex = 380)

107.84 12.75

Carbonyl content (nmol/mg protein) 3.1 ± 0.4 20.24 ± 1.2b

Nitrotyrosine conc. (nmol/mg protein) – 29.6 ± 2.3c

Dityrosine conc. (nmol/mg protein) – 112.7 ± 3.1c

Far-UV CDdMRE 222 nm −21,606 −15,971e�-Helix (%) 64 45

Near-UV CDdMRE 275 nm –1263 –1072

FTIR, peak positions(Amide I, cm−1) 1656 1638(Amide II, cm−1) 1553 1546

a �ex: excitation wavelength.b p < 0.001 versus native HSA.c p < 0.001.d MRE value in deg cm2 dmol−1.e �-Helix (%) calculated by Chen et al. method.

Direct binding ELISA of (A) native HSA and (B) peroxynitrite-modified HSA withpre-immune (-�-) and immune (-�-) sera. Microtitre plates were coated with therespective antigens (10 �g/ml).

This clearly indicates that peroxynitrite modification of HSA hasmade the protein an even more potent immunogen which mightbe due to the generation of neo-epitopes on the protein molecule.Preimmune serum when used as control showed negligible bindingwith either immunogen under identical experimental conditions.The induced anti-peroxynitrite-modified HSA antibodies were alsoexamined for their binding to denatured HSA to check the speci-ficity of these antibodies towards HSA structure. Very weak bindingof these antibodies to denatured HSA confirmed their specificitytowards the tertiary structure of protein (data not shown).

IgG were purified from preimmune and immune rabbit antis-era of native and peroxynitrite-modified HSA by Protein A-agaroseaffinity chromatography. The purified IgG eluted as a single sym-metrical peak and its purity was confirmed by the presence of asingle homogeneous band by SDS–PAGE under non-reducing con-ditions (data not shown). Anti-peroxynitrite-modified-HSA IgG andanti-native-HSA IgG showed strong binding with their respectiveimmunogens as determined by direct binding ELISA. However, pre-immune IgG showed negligible binding under identical conditions(data not shown).

Competitive inhibition ELISA was done to detect the specificityof experimentally produced antibodies against peroxynitrite-modified HSA. An inhibition of 55.3% was observed when nativeHSA was used as inhibitor of anti-peroxynitrite-modified-HSA IgG,

P. Ahmad et al. / International Journal of Biological Macromolecules 59 (2013) 349– 356 355

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Fig. 9. Band shift assay of anti-peroxynitrite-modified-HSA IgG binding to (A) nativeHSA and (B) peroxynitrite-modified HSA. Electrophoresis was performed on 10%SDS–PAGE for 4 h at 80 V. (A) Native HSA (10 �g) was incubated with 0, 10, 20, 30,40 and 50 �g anti-peroxynitrite-modified-HSA IgG, respectively (lanes 1–6) for 2 h

ig. 8. Inhibition ELISA of anti-peroxynitrite-modified-HSA IgG. The inhibitors wereative HSA (-�-) and peroxynitrite-modified HSA (-�-). Microtitre plates wereoated with peroxynitrite-modified HSA (10 �g/ml).

hile it was 83.2% when peroxynitrite-modified HSA was useds inhibitor (Fig. 8). Fifty percent inhibition was achieved at annhibitor (peroxynitrite-modified HSA) concentration of 3.9 �g/ml.he enhanced immunogenicity of HSA after peroxynitrite modi-cation is probably the result of the generation of neo-epitopesn HSA molecule consequent to modification. The generation ofhese neo-epitopes might be the result of nitration and oxidationf HSA. Therefore, it appears that two types of antibodies werenduced; one recognizes neo-epitopes while the other recognizesld epitopes. Furthermore, cross-reactivity of anti-peroxynitrite-odified-HSA IgG with different inhibitors was checked to confirm

hat the induced antibodies were highly specific for peroxynitrite-odified HSA. Results of cross-reactivity with different inhibitors

ave been summarized in Table 2.Band shift assay was performed to visualize the speci-

city of antibodies and to confirm the interaction of nativend peroxynitrite-modified HSA with the isolated IgG. Thentigen–antibody complexes were prepared by incubating a con-tant amount of native and peroxynitrite-modified HSA withncreasing concentrations of anti-peroxynitrite-modified-HSA IgGnd then electrophoresing these immune complexes on 10%DS–PAGE. The gel pattern showed that with increasing amountf anti-peroxynitrite-modified-HSA IgG, there was correspondingncrease in the formation of high molecular mass immune com-

lexes (Fig. 9). The free antigen intensity was decreased showing

ts greater involvement in the formation of immune complexes.owever, there was slight shifting in the mobility when native HSAas used instead of peroxynitrite-modified HSA under identical

able 2ross-reaction of anti-peroxynitrite-modified-HSA IgG with different inhibitors.

Inhibitors % Inhibitionmax at 20 �g/ml

Peroxynitrite-modified HSA 83.2Native HSA 55.3Peroxynitrite-modified IgG 64.4Native IgG 42.3Peroxynitrite-modified H2A histone 59.4Native H2A histone 37.2Peroxynitrite-modified tyrosine 65Native tyrosine 32.6Peroxynitrite-modified tryptophan 34.4Native tryptophan 17Peroxynitrite-modified phenylalanine 22.6Native phenylalanine 15.2

at 37 ◦C and overnight at 4 ◦C. (B) Peroxynitrite-modified HSA (10 �g) was incubatedwith 0, 10, 20, 30, 40 and 50 �g anti-peroxynitrite-modified-HSA IgG respectively(lanes 1–6) under identical conditions.

experimental conditions. These results indicate that while nativeHSA binds to the anti-peroxynitrite-modified-HSA IgG to someextent due to the presence of old epitopes, the binding is moreprominent with modified protein. This shows that modified HSAhas got both the epitopes, i.e., old and neo-epitopes formed byperoxynitrite modification. This confirms the specificity of anti-peroxynitrite-modified-HSA IgG.

Since HSA is one of the most important plasma proteins withmultiple functions, therefore, its structural stability becomes themain factor to carry out all its functions properly. Our results clearlydemonstrate that in vitro modification of HSA with peroxynitritebrings about biophysical and biochemical alterations in the protein,which was confirmed by the generation of highly specific antibod-ies in experimental animal. Antibodies against modified/damagedHSA have been reported in various diseases like diabetes mel-litus, systemic lupus erythematosus (SLE), cancer, etc. [35,36].Our results suggest that structurally perturbed HSA exhibits neo-epitopes which are not ordinarily present on the native molecule.We propose that these epitopes project the physiologic protein as‘alien’ for the immune system leading to antibody generation invarious diseases.

Acknowledgements

The assistance from the Institution (AMU) as well as infrastruc-tural support from DST-FIST facility of the Department is thankfullyacknowledged. The authors are grateful to Dr. Shabbir Ahmad,

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epartment of Physics, and Mr. Gulam Rabbani, Interdisciplinaryiotechnology Unit, AMU, Aligarh (India) for providing instrumen-al facilities and analysis of the results. Encouragement and helpuring the course of work by Dr. Kiran Dixit is duly acknowledged.

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