Spectroscopic investigation of the interaction of the toxicant, 2-naphthylamine, with bovine serum albumin

J BIOCHEM MOLECULAR TOXICOLOGYVolume 25, Number 6, 2011

Spectroscopic Investigation of the Interaction of theToxicant, 2-Naphthylamine, with Bovine Serum AlbuminYan Liu,1 Mingmao Chen,2 Guangling Bian,1 Junfeng Liu,1 and Ling Song1

1The State Key Lab of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou,Fujian 350002, People’s Republic of China; E-mail: [email protected] of Biomaterials & Artificial Organ, Institute of Biomedical Engineering, Chinese Academy of Medical Science & Peking UnionMedical College, Key Laboratory of Biomedical Material of Tianjin, Tianjin 300192, People’s Republic of China

Received 20 March 2011; revised 6 May 2011; accepted 30 May 2011

ABSTRACT: The mechanism of interaction betweenbovine serum albumin (BSA) and 2-naphthylamine(2-NA) in aqueous solution was investigated by fluo-rescence spectroscopy, circular dichroism (CD) spectra,and UV–vis spectroscopy. It was proved from fluores-cence spectra that the fluorescence quenching of BSAby 2-NA was a result of the formation of complex be-tween 2-NA and BSA, and the binding constants (Ka)as well as the numbers of binding sites for 2-NA inBSA were determined according to the modified Stern–Volmer equation. The results of synchronous fluores-cence and CD spectra demonstrated 2-NA could de-crease the amount of α-helix of BSA, leading to theloosening of protein skeleton. UV–vis spectroscopyand resonance light scattering spectra (RLS) results alsosuggested the conformation of BSA were changed andthe BSA aggregation occured, which could induce toxiceffects on the organism. C© 2011 Wiley Periodicals, Inc.J Biochem Mol Toxicol 25:362–368, 2011; View this articleonline at wileyonlinelibrary.com. DOI 10:1002/jbt.20400

KEYWORDS: 2-Naphthylamine; Bovine serum albumin;Protein denaturation; Carcinogen

INTRODUCTION

There are presently more than 1 billion smokersworldwide and smoking is the number one preventablecause of death according to the WHO. 2-naphthylamine(2-NA), an aromatic primary amine (structure for-mula shown in Figure 1) is a well-known carcinogen(LD50 = 727 mg/kg in rat) found in amounts of 1.0–

Correspondence to: Ling Song.Contract Grant Sponsor: National Basic Research Program of

China.Contract Grant Number: 2010CB933501.

c© 2011 Wiley Periodicals, Inc.

20 ng/cigarette in cigarette smoke [1–3]. Like mosttoxic substances, 2-NA could infiltrate into organismsthrough the respiratory tract and skin and then dam-ages tissues and organs owing to its interactions withbiological macromolecules. Previous studies indicated2-NA exhibited clearly genotoxic in vivo [4]. Epidemi-ological studies have shown that occupational expo-sure to 2-NA, either alone or present as an impurity inother compounds, causes bladder cancer [5–7]. Stud-ies of dyestuff workers and of chemical workers ex-posed mainly to 2-NA found increased risks of bladdercancer [6–8]. When administered orally, 2-NA causedmalignant bladder tumors in hamsters, dogs, and rhe-sus monkeys and liver tumors in mice; bladder tumorswere also observed in rats at a low incidence (IARC1987). It is reported that 2-NA is also able to inhibitbrain mouse MAO A and B in vitro [1].

Protein denaturation often reflected in the destruc-tion of the molecular conformation by environmentalfactors such as temperature, pH, ionic strength, tox-ins, and so on [9]. These factors could change thenative conformation of biomacromolecules, leadingto the structural damage of protein skeleton. Dena-tured proteins can result in illness or even death. Infact protein denaturation is linked to many diseasessuch as prion encephalopathies, cataract, Alzheimer’sdisease, Parkinson’s diseases, and dementias [10–13].Among biomacromolecules, serum albumin (SA) playsan important role in maintaining the osmotic pressureneeded for proper distribution of body fluids betweenintravascular compartments and body tissues [14]. Italso acts as a plasma carrier by nonspecifically bindingvarious exogenous and endogenous ligands. It can in-crease the apparent solubility of hydrophobic drugs inplasma and modulate their delivery to cell in vivo andin vitro [15,16]. Interaction with albumin could be crit-ical for understa nding the toxicity of toxic substancesand its distribution in the organism. It has been an in-teresting research field in life science, chemistry, and

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Volume 25, Number 6, 2011 TOXIC EFFECT OF 2-NAPHTHYLAMINE ON BSA 363

FIGURE 1. Emission spectra of BSA in the presence of 2-NA ofvarious concentrations (T = 292 K, λex = 280nm). c(BSA) = 1.0 × 10−5

mol L−1; c(2-NA)/(10−5 mol L−1), A–H: 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4,respectively.

clinical medicine. Although there are lots of studies onthe interaction of small molecules and bovine serum al-bumin (BSA), research on the interaction between BSAand 2-NA which has the potential carcinogenicity hasnot been reported. Therefore, it is necessary to eluci-date reaction mechanism and the toxicological actionof 2-NA to the organisms at molecular level.

In this paper, BSA was selected as our studyprotein model because of its low cost, ready availabil-ity, unusual ligand binding properties, medical impor-tance, and particularly its structural homology with hu-man SA [17]. The interaction between BSA and 2-NAwas studied under physiological conditions by fluo-rescence, UV–vis spectroscopy, resonance light scatter-ing spectra (RLS), and circular dichroism (CD) spec-troscopy. Many attempts were made to investigate thebinding mechanism, the special binding site, and theeffect of 2-NA on the conformational changes of BSA.We hope that this work can elucidate the propertiesof 2-NA-protein complex and dangerous mechanismof 2-NA in organism, and its toxicological action atmolecular level.

MATERIALS AND METHODS

Apparatus

Fluorescence data were obtained on a Cary EclipseSpectrofluorimeter (Varian, Walnut Creek, USA)equipped with 1.0 cm quartz cells. Absorption spec-tra were recorded on a Lambda-35 spectrophotometer(PerkinElmer, Waltham, USA) equipped with 1.0 cm

quartz cells at room temperature. CD spectra weremeasured on a MOS-450/AF-CD Spectropolarimeter(Bio-Logic, Claix, France) at room temperature underconstant nitrogen flush. All pH measurements weremade with a PHS-3C acidity meter.

Reagents

A 2-NA stock solution (1.0 × 10−3 mol L−1) was pre-pared by diluting 2-NA in a 100 mL volumetric flask.The BSA working solutions were prepared to be theconcentration of 1.0 × 10−3 mol L−1 in phosphate buffersolutions (pH 7.40) and stored in the dark prior to use.The solutions were stored at 4◦C and shaken gentlyas needed to redissolve the contents. All the chemicalswere of analytical purity, and doubly distilled waterwas used throughout the experiment.

Experimental Methods

Fluorescence Spectra

To prepare the test solutions, 1.0 mL of BSA so-lution and 1.0 mL of phosphate buffer solution wereplaced in a 10 mL standard volumetric flask. A def-inited volume of 2-NA solution was added, and themixture was diluted to the certain volume with phos-phate buffer and mixed. After preparation, the reactionsystems were incubated for 30 min, then the spectrawere measured on the Cary Eclipse fluorescence spec-trophotometer.

Fluorescence emission spectra were recorded at292 K in the range of 300–450 nm. The width of theexcitation and emission slit was set to 10.0 and 2.5 nm,respectively. An excitation wavelength of 280 nm waschosen and the scanning speed was 600 nm/min. Allthe experiments were conducted in triplicate.

Synchronous Fluorescence Spectra

Synchronous fluorescence spectra of solutions pre-pared as described above were measured on the CaryEclipse fluorescence spectrophotometer. The excitationwavelength (λex) was at 280 nm. The excitation andemission slit widths were set at 10.0 and 2.5 nm. TheD-value (�λ) between the excitation and emissionwavelengths was set at 15 or 60 nm.

Resonance Light Scattering Spectra

RLS were also recorded on the Cary Eclipse fluo-rescence spectrophotometer at 292 K within the wave-length range of 200–700 nm. The width of the exci-tation and emission slits was set to 10.0 and 2.5 nm,

J Biochem Molecular Toxicology DOI 10:1002/jbt

364 LIU ET AL. Volume 25, Number 6, 2011

respectively. The concentration of BSA was kept at 1.0× 10−5 mol L−1, and then 2-NA was gradually addedto the system with different amount.

Absorption Spectra

UV–visible absorption spectra were measured on aLambda-35 spectrophotometer. All absorption spectraused phosphate buffer solution (pH 7.40) as the refer-ence solution.

Circular Dichroism Spectra

Circular dichroism spectra were measured on aMOS-450/AF-CD Spectropolarimeter at room temper-ature under constant nitrogen flush over a wavelengthrange of 260–200 nm. Quartz cells have path length andvolume of 0.1 cm and 400 μL, respectively. The scan-ning speed was set at 100 nm min−1. Each spectrumwas the average of three successive scans and appro-priate buffer solutions running under the same condi-tions were taken as blank and their contributions weresubtracted from the experimental spectra. The concen-tration of BSA was kept at 1.0 × 10−5 mol L−1 and themolar ratio of BSA to 2-NA was varied from 1:0 to 1:1.5.The contents of different secondary structures of BSAwere analyzed by the algorithm SELCON3 [18].

RESULTS

Fluorescence Measurement

The interaction of 2-NA with BSA was studiedby measuring the change of the intrinsic fluorescenceat 350 nm after excitation at 280 nm wavelength,which can effectively excite the tyrosine and trypto-phan residues of BSA [19]. Figure 1 shows the fluores-cence emission spectra of BSA in the presence of 2-NAwith a peak at 350 nm which indicated that specific ty-rosines and tryptophans of BSA are partly exposed tothe solvent. With the increasing concentration of 2-NA,the fluorescence intensity of BSA decreased regularly,indicating that interactions between 2-NA and BSA oc-curred and 2-NA-BSA complex may form. In addition,the fluorescence emission intensity of 2-NA was veryweak at 350 nm at the same condition although thereis a strong absorption at 410 nm (see inset in Figure 1),which indicated that the effect of 2-NA on the fluores-cence intensity of BSA at around 350 nm could be negli-gible. Generally speaking, the fluorescence quenchingtypes often include static and dynamic quenching [20].In order to obtain a clear insight into the quenchingmechanism, the fluorescence quenching data are ana-

lyzed by the Stern–Volmer equation [21]:

F0/F = 1 + KSV[Q] = 1 + kqT0[Q] (1)

where F0 and F are the steady-state fluorescence in-tensities in the absence and presence of quencher, re-spectively. KSV is the Stern–Volmer quenching constant,[Q] is the concentration of quencher, kq is the quench-ing rate constant of the biomolecule and the value ofthe maximum scattering collision quenching constant is2.0 × 1010 L mol−1 s−1 [22]. τ0 is the average fluorescencelifetime of biomolecular and equal to 10−8s [21].

The fluorescence spectra of BSA in the presenceof different amount of 2-NA were shown in Figure 1.In order to identify the type of quenching originatedfrom the interaction of 2-NA and BSA, the fluores-cence quenching data were analyzed using the Stern–Volmer equation (Eq. (1)). Using Eq. (1), KSV was de-termined by linear regression of a plot of F0/F against[Q]. The Stern–Volmer plots are shown in Figure 2a. The

FIGURE 2. (a) Stern–Volmer plots for the quenching of BSA by2-NA at 292 K; (b) Modified Stern–Volmer plots for the quenchingof BSA by 2-NA at 292 K. Conditions: c(BSA) = 1.0 × 10−5 mol L−1,c(PBS) = 5 × 10−2 mol L−1 (pH 7.40)



quenching constants were Ksv = 2.67 × 104 L mol−1 andKq = 2.67 × 1012 L mol−1 s−1. Since Kq is greater than themaximum dynamic quenching constant (2.0 × 1010 Lmol−1 s−1), the probable quenching mechanism is staticquenching, which indicates that there are interactionsbetween 2-NA and BSA, generating a stable complex.

Numbers of Binding Sites

For static quenching, the following equation wasemployed to calculate the binding constant and bindingsites at different concentration of 2-NA [21].

log(F0 − F )/F = logKA + n log[Q] (2)

F0 and F are the fluorescence intensities of BSA in theabsence and presence of 2-NA, KA is the binding con-stant, and n is the number of binding sites. As Figure 2bshows, a plot of log (F0 – F )/F versus log [Q] yieldedKA and n values of 9.55 × 103 and 0.909, respectively.The value of n approximately equals to 1 suggestedthat there was one type of binding sites for 2-NA to-ward BSA and the most likely binding site is Site Ihydrophobic cavity.

Synchronous Fluorescence Spectroscopy

To further demonstrate the interaction betweenBSA and 2-NA, we used synchronous fluorescencespectroscopy, which can provide information about themolecular environment in the vicinity of the chromo-sphere molecules and the changes of maximum emis-sion wavelength reflect the conformational changes ofBSA. Synchronous fluorescence spectra were obtainedby simultaneously scanning excitation and emissionmonochromators. When �λ between excitation wave-length and mission wavelength is 15 or 60 nm, the syn-chronous fluorescence offers characteristics of tyrosineresidues or tryptophan residues [23]. Synchronous flu-orescence spectra of BSA upon addition of 2-NA at�λ = 15 and 60 nm are shown in Figure 3. The maxi-mum emission wavelength had a slight red shift when�λ = 60 nm, which suggested a less hydrophobic en-vironment around the tryptophan residues. The reasonwas likely that the hydrophobic amino acid structuresurrounding tryptophan residues in BSA tends to col-lapse slightly, resulting in tryptophan residues moreexposed to the aqueous phase. When �λ is equal to15 nm, the maximum emission wavelength of tyrosineresidues nearly has no any shift, indicating that therewas no change in the microenvironment of the tyrosineresidues.

FIGURE 3. Synchronous fluorescence spectrum of BSA: (a) �λ = 15nm; (b) �λ = 60 nm. c(BSA) = 1.0 × 10−5 mol L−1; c(2-NA)/(10−5 molL−1), A-I: 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.5, respectively.

Resonance Light Scattering Spectra of BSAin the Presence of 2-NA

The RLS measurement was carried out for con-firming the quenching process (Figure 4). The RLS re-sponses of free BSA without 2-NA was weak, whilethe intensity was enhanced remarkably with increas-ing 2-NA added into the system. The production of in-creasing RLS indicated that 2-NA might interact withBSA and assembled around BSA, then a larger size of2-NA-BSA aggregate was formed.

Absorption Spectroscopy of BSA in thePresence of 2-NA

The UV absorption of protein is mainly due to theelectronic excitation of aromatic amino acids, such astryptophan, tyrosine, phenylalanine, and histidine [24].The varying solution conditions of these chromophores



FIGURE 4. The RLS spectra of BSA-2-NA systems. c(BSA) = 1.0 ×10−5 mol L−1; c(2-NA)/(10−5 mol L−1), A–F: 0, 0.5, 1.0, 1.5, 2.0, 2.5,respectively.

cause changes in the microenvironment, which are re-flected in the different UV–vis spectra. The absorp-tion peak of BSA was at about 218 nm and 280 nm,which contributed to π → π* transfer for the peptidebond and aromatic amino acid, respectively. The ab-sorption spectrum of BSA was observed to change ob-viously when 2-NA was added (Figure 5a). The max-imum absorption of BSA decreased dramatically andthe peak at 218 nm became flat gradually with increas-ing concentrations of 2-NA. This indicated that BSAformed groundstate complexes with BSA and inducedchanges in protein conformation, which caused observ-able changes in the absorption spectra.

Circular Dichroism Spectra of BSAin the Presence of 2-NA

The CD experiments were carried out at room tem-perature to make the study of conformational changeof BSA more reliable and scientific. The CD spectra ofBSA exhibited a signal characteristic of α-helix struc-ture with two negative bands in the far-UV region at208 and 222 nm [25]. The reasonable explanation is thatthe negative peaks between 208 and 222 nm are bothcontributed to n→ π* transfer for the peptide bond ofα-helix [26]. The CD spectra of the 2-NA–BSA system atpH 7.4 are shown in Figure 5b and are analyzed by thealgorithm SELCON3. The fraction contents of α-helix,β-turn, β-sheet, and random forms for BSA in the ab-sence and presence of 2-NA are shown in Table 1. The α-helix of the secondary structure of BSA decreased andthe β-sheet, β-turn, and unordered structure increasedwith the addition of 2-NA at BSA/2-NA ratios from1:0 to 1:1.5. It is also clear that α-helical content (thetotal content of regular and distorted α-helix) differed

FIGURE 5. (a) The UV–vis spectra of BSA with different concentra-tion of 2-NA.c(BSA) = 1.0 × 10−5 mol L−1; c(2-NA)/(10−5 mol L−1),A–H: 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, respectively; (b) The CD spectraof the 2-NA–BSA system at pH 7.4, c(BSA) = 1.0 × 10−5 mol L−1;c(2-NA)/(10−5 mol L−1), A–F: 0, 0.2, 0.4, 0.8, 1.2, 1.5, respectively.

from 57.3% to 44.8%, implying that 2-NA bound withthe amino acid residues of the main polypeptide chainof protein and destroyed their hydrogen bonding net-works. Furthermore, a degree in reduction in the per-centage of the regular α-helix (22.6%) and an increasein the distorted α-helix (10.1%) were observed, whichsuggested that the binding of 2-NA to BSA induced alittle unfolding of the polypeptides of protein, whichresults in the exposure of some hydrophobic regionsthat were previously buried increasing as mentionedabove.

DISCUSSION

Fluorescence, CD, RLS, and UV–vis absorptionspectroscopic approaches were applied to investi-gate the interaction between BSA and 2-NA under



TABLE 1. Fractions of Different Secondary Structures Determined by SELCON3

Molar Ratio

[BSA]/[2-NA] H(r) (%) H(d) (%) S(r) (%) S(d) (%) Tr (%) Un (%)

1/0 33.0 24.3 3.7 3.1 18.2 17.71/0.4 32.1 24.1 3.3 2.8 18.8 18.91/0.8 31.9 23.1 3.0 2.7 18.0 21.31/1.5 10.4 34.4 22.2 13.6 8.1 11.3

H(r) regular α-helix; H(d) distorted α-helix; S(r) regular β-strand; S(d) distorted β-strand; Tr turns; Un unordered structure.

simulative physiological conditions. Fluorescence dataimplied that 2-NA was a strong quencher and boundto BSA with static quenching process. The results ofsynchronous fluorescence spectra suggested that thereis one type of binding site for 2-NA in BSA, and a stablecomplex is generated. CD results demonstrated that 2-NA decreases the amount of α-helix and promotes theunfolding process, leading to a loosening of the proteinskeleton. The absorption decrease and flat peak phe-nomenon from the ultraviolet absorption spectra, aswell as the decrease of the helicity from the CD, agreethat 2-NA has toxic effects that change the secondarystructure of BSA. In summary, 2-NA changes the mi-croenvironment of BSA and its secondary structure,and the toxic effects are strengthened with the dose of2-NA increase.

ACKNOWLEDGMENTS

The authors would like to thank State Key Labof Structural Chemistry, Fujian Institute of Researchon the Structure of Matter, Chinese Academy of Sci-ences and the Scientific Research Foundation for theReturned Overseas Chinese Scholars, State EducationMinistry.

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Spectroscopic investigation of the interaction of the toxicant, 2-naphthylamine, with bovine serum albumin