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aDepartment of Chemistry and Bioinforma

Biology, 4, Raja S.C. Mullick Road, Ko

banerji@gmail.com; Fax: +91 33 24735197bDepartment of Structural Biology and B

Chemical Biology, 4, Raja S.C. Mullick Road

Cite this: RSC Adv., 2014, 4, 22536

Received 24th March 2014Accepted 24th April 2014

DOI: 10.1039/c4ra02569e

www.rsc.org/advances

22536 | RSC Adv., 2014, 4, 22536–2254

Binding of hemoglobin to ultrafine carbonnanoparticles: a spectroscopic insight into a majorhealth hazard

Biswadip Banerji,*a Sumit Kumar Pramanik,a Uttam Palb and Nakul Chandra Maitib

Carbon nanoparticles (CNPs) are light and easily absorb into different parts of organs of the human body.

They are suspended particulate matters of respirable sizes. In the atmosphere, ultrafine CNPs are known

to be generated mainly from the combustion of fuels and have been reported to be a major contributor

to the induction of cardiopulmonary diseases. In third world countries, these diseases are more prevalent

because of the higher abundance of ultrafine CNPs in the air. Different nanostructured materials, when

exposed to the human body, can easily enter into the body through the lungs or other organs and

tissues. In the laboratory, ultrafine carbon nanoparticles were synthesized and their structure was

confirmed by DLS experiments, TEM and AFM imaging studies. Their interactions with hemoglobin (Hb)

and myoglobin (Mb) were studied using fluorescence spectroscopy. The results indicate a remarkably

strong interaction between carbon nanoparticles and Hb (or Mb). Temperature dependent steady state

fluorescence spectroscopy showed exothermic binding of Hb to CNPs, which is favored by enthalpy and

entropy changes. A circular dichroism study also indicated significant change in the protein secondary

structure and a partial unfolding of the helical conformation. These findings are highly important for

understanding the interactions between CNPs and Hb (or Mb), which might help to better clarify the

potential risks and undesirable health hazards associated with carbon nanoparticles.

Introduction

The domain of nanotechnology is rapidly increasing and todaythere are more than 1500 nanoproducts in the market.1,2 Thedifferent nanostructured materials such as nanotubes,3 nano-particles,2 nanocages,4 nanopowders,5 nanowires,6 nanoshells,7

nanorods,8 nanobers,9 quantum dots,10 fullerenes,11 lipo-somes,12 neosomes,13 nanoclusters,14 nanomeshes,15 nano-crystals,16 nanolms17 and nanocomposites18 are internationallyproduced in bulk quantities because of their wide potentialapplications in skincare and consumer products, healthcare,photonics, electronics, biotechnology, engineering products,pharmaceuticals, drug delivery, agriculture, etc. When differentnanostructured materials are exposed to the human body theycan easily enter into the body through the lungs or other organsand tissues such as the brain, liver, kidney, heart, colon, boneand blood via food, drink and medicine.19–22 These nano-materials may cause cytotoxic effects, e.g., the deformation andinhibition of cell growth leading to various diseases in humansand animals. The toxicity and interactions of these

tics, CSIR-Indian Institute of Chemical

lkata, India-700032. E-mail: biswadip.

, 91 33 24723967; Tel: +91 33 24995709

ioinformatics, CSIR-Indian Institute of

, Kolkata, India-700032

1

nanomaterials with biological systems largely depend on theirphysicochemical properties such as size, concentration, solu-bility, chemical composition and stability.23–26

Carbon nanoparticles (CNPs) are popular nanomaterialsbecause of their interesting properties, which are desirable inmany industrial purposes.27 CNPs have physicochemical prop-erties that are already in use in the commercial, environmentaland medical sectors.28–32 However, with diameters in the nano-scale range these extremely small particles may constitute ahealth risk.33

Particle emission studies from a steady burning singleparaffin wax candle found the diameter of the produced parti-cles to be around 30 nm.34 The concentrations of the particlesemitted from a pure wax candle and a scented candle in achamber were reported to be 240 000 particles per cm3 and69 000 particles per cm3, respectively.35 CNPs are very light andeasily enter in the working environment as suspended partic-ulate matter of respirable sizes.36 Therefore, these CNPs couldpose an occupational inhalation exposure hazard. CNPs of sizes<2.5 micron are found in the combustion streams of methane,propane, butane and natural gas ames of typical stoves. Bothindoor and outdoor ne particle samples contain a signicantfraction of CNPs. It has already been reported that CNPs aremore toxic than quartz and cause occupational health hazard ifchronically inhaled.37 Environmental ne CNPs are known to begenerated mainly from the combustion of fuels, and has been

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reported to be a major contributor to the induction of cardio-pulmonary diseases caused by pollutants.38 Therefore, CNPsfrom manufactured and combustion sources in the environ-ment could have adverse effects on human health.

Carbon nanotubes (CNTs) are known to have a wide range ofbiomedical applications but the importance of nanoparticle–protein interactions are not stressed enough,39 althoughsystemic interactions of CNTs with various plasma proteinshave been reported. However, despite the fact that CNPs canpenetrate the plasma membrane, knowledge regarding theeffect of CNPs on blood corpuscular protein, i.e. haemoglobin,is still obscure.40–44 Inhalation of CNPs from the environmentdirectly exposes them to our blood circulatory system.42–44

Therefore, knowledge of the interaction between CNP and theoxygen transporter protein, hemoglobin, is essential to clarifythe impact of CNPs on human health. In the current paper wepresent a detailed study on the interaction of hemoglobin withCNPs, using uorescence spectroscopy and circular dichroismspectroscopy (CD). For a better understanding, we have usedmyoglobin alongside hemoglobin, as this can be considered asa monomeric unit or hemoglobin. Myoglobin is functionallysimilar to hemoglobin and shares a similar structural fold,despite the dissimilarities in amino acid composition, thus, itprovides a deeper insight into the effect of CNPs onhemoglobin.

Materials and methodsMaterials

Human hemoglobin and myoglobin were purchased fromSigma-Aldrich (USA). Carbon soot, acetone, TrisHCl and nitricacid were also purchased from Sigma-Aldrich. All the sampleswere prepared in 20 mM Tris–HCl buffer of pH 7.4 for CNP–protein interaction studies. Deionized and triple distilled waterwas used for preparing a buffer solution that was passedthrough 0.22 mm pore size Millipore lters (Millipore India Pvt.Ltd., Bangalore, India). A carbon coated copper grid for the TEMstudy was purchased from Allied Scientic Product, USA. ASTMV1 Grade Ruby Mica sheet for AFM study was purchased fromMicafab India Pvt. Ltd., Chennai, India.

Synthesis of carbon nanoparticles (CNPs)45

100 mg carbon soot was added to 60 mL of 6 M Nitric acid in a25 mL three necked ask. The reaction mixture was thenreuxed at 100 �C for 10 hours under stirring. Aerwards, ablack solution was obtained. The solution was then centrifugedat 2000 rpm for 15minutes to separate out the unreacted carbonsoot. Subsequently, the aqueous solution was mixed withacetone and then centrifuged at 30 000 rpm for 20 minutes anda black precipitate was obtained. This precipitate was washed in5–10 mL water to remove the excess nitric acid. Then, theprecipitate was dried and its weight was measured. This wasthen dissolved in an appropriate amount of buffer to obtain thedesired concentration. The pH of the nal suspension wasadjusted to 7.4 and this suspension was used for furtherexperiments.

This journal is © The Royal Society of Chemistry 2014

Dynamic light scattering (DLS)-based zeta-potentialmeasurements

To determine the size distribution of the CNPs, DLS experi-ments (model: Zetasizer Nano Z, Malvern Instruments Ltd,United Kingdom) were carried out with the aqueous suspen-sion. The scattered lights were collected at a 90� angle. Datawere acquired and analyzed by a Precision Deconvolve program.For a typical DLS experiment, 200 mL of a sample solution wasslowly pipetted into a clean quartz micro-cuvette.

TEM sample preparation and imaging

For TEM imaging, 20 mL of the nanoparticle solution was placedon a 300-mesh carbon coated copper grid and the excesssamples were removed cautiously by a tissue paper. This wasnally dried and the images of the resulting nanoparticlesolutions were recorded on a Tecnai G2 Spirit Bio TWIN (Type:FP5018/40) at an acceleration voltage of 80 kV.

AFM sample preparation and imaging45

Twenty microliters of the nanoparticle solution was depositedonto a freshly cleavedmuscovite Rubymica sheet for 5 to 10min.Aer 10min, the samplewasdriedusinga vacuumdryer. AnAAC-mode atomic force microscopy was performed using a Pico Plus5500 AFM (Agilent Technologies, Inc., Santa Clara, CA, USA) witha piezo scanner at a maximum range of 9 mm. Microfabricatedsilicon cantilevers of 225 mm in length with a nominal springforce constant of 21–98 Nm�1 were used from nanosensors. Thecantilever oscillation frequency was tuned to the resonancefrequency. The cantilever resonance frequencywas 150–300 kHz.The images (512 � 512 pixels) were captured with a scan sizebetween 0.5 and 5 mmat a scan speed rate of 0.5 rpm. The imageswere processed by attening using Pico view soware (MolecularImaging Inc., Ann Arbor, MI, USA). The image presented in thispaper was derived from the original data. Length, height andwidth were measured manually using Pico view soware.

Fluorescence spectroscopy

The steady-state uorescence spectra were recorded with aPerkin Elmer LS-45 spectrouorophotometer at 25 �C. Thesamples were excited at 280 nm wavelength and the emissionspectra were recorded over the emission range of 300–450 nm.In all the cases, the excitation and emission slit widths weremaintained at 5 nm each. The intrinsic uorescence of proteinwas measured in the presence and the absence of the CNPs.Intrinsic uorescence of Mb and Hb originated from both theTrp and Tyr. Hb contains 6 Trp and 12 Tyr whereas Mb contains2 Trp and 2 Tyr. The uorescence of the protein was quenchedin the presence of the CNPs. The quenching experiment wascarried out simply by adding small aliquots of concentratedCNP solution to the hemoglobin or myoglobin solution taken ina 1 cm path length quartz cuvette. The protein concentrationwas maintained at 0.5 mM and the CNP concentration wasvaried from 0.5 mM to 5 mM. The optical density of the solutionat the excitation wavelength was kept below 0.05. Small errorsdue to dilution upon the addition of the CNPs were neglected.

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Fluorescence intensities at the emission maximum were recor-ded as a function of ligand concentration. To derive the bindingparameters, the obtained data were analyzed using a modiedStern–Volmer equation (eqn (1)).

F0/DF ¼ 1/fK[Q] + 1/f (1)

whereF0 is theuorescence intensity in theabsenceof anexternalquencher,DF is the difference in uorescence in the absence andin the presence of the quencher at concentration [Q], K is theStern–Volmer quenching constant and f is a fraction of the initialuorescence which is accessible to the quencher. The bindingdissociation constant, Kd, was measured as the reciprocal of K.

Temperature dependent uorescence quenching was per-formed to obtain the Kd values as a function of temperature, andthen the thermodynamic parameters of binding were deter-mined from it bytting van'tHoff's equation (eqn (2)) to thedata.

ln K ¼ �DH�/RT + DH�/R (2)

where K is the equilibrium constant (here the Stern–Volmerquenching constant) of binding at a corresponding temperatureT and R is the gas constant. The equation gives the standardenthalpy change (DH�) and standard entropy change (DS�) onbinding. The free energy change (DG�) has been estimated fromthe following relationship (eqn (3)):

DG� ¼ DH� � TDS� (3)

CD spectroscopy

The conformational changes in the secondary and tertiarystructures of hemoglobin and myoglobin on the binding ofCNPs were studied using a Jasco J-810 spectrometer at 25 �C.The CD spectra of Hb andMb were nally obtained by averagingve successive scans recorded at a scan speed of 50 nm min�1

and subtracting the appropriate blanks (tris buffer) from thesespectra. For Hb and Mb proteins, the changes in the far UV-CDspectra (200–250 nm) provide insights about the changes in thesecondary structure of the proteins. The protein concentrationswere 0.75 mM for Hb and 3.0 mM for Mb. The results areexpressed in terms of mean residual ellipticity (MRE) in � cm2

dmol�1 according to the equation given below.46

Fig. 1 TEM (A) and AFM (B) image of synthesized ultrafine CNPs.

22538 | RSC Adv., 2014, 4, 22536–22541

MRE ¼ observed CD ðm degÞCpnl � 10

where Cp is the molar concentration of the protein, n is thenumber of amino acid residues (n¼ 574 for Hb and 154 for Mb)and l is the path length (here l ¼ 0.1 cm, i.e. the path length ofthe cuvette used). The helical content of free and bound proteinwas nally evaluated from the MRE values at 208 nm using theequation given below.46

a-helix % ¼��MRE208 � 4000

33 000� 4000

�� 100

Results and discussionDLS study

The results of the DLS experiment showed that the meanparticle size of the CNPs is 20.17 nm. The zeta potentialdistribution of CNPs are negative charged to�39.1 mV in water,which is sufficient to keep the particles from interacting witheach other; therefore, maintains a stable particle size of thesample. The resulting negative charges of CNPs that have beentagged are attributed to the negative surface charge on them.

TEM and AFM imaging

The transmission electron microscopy (TEM) images of theprepared CNPs have a spherical morphology with a relativelyregular diameter of �20.0 nm (Fig. 1A). They are almostuniform in nature. The atomic force microscopy (AFM) studywas in good agreement with the TEM results, which showed thatthe average sizes have a spherical morphology with a relativelyregular diameter of �22.0 nm (Fig. 1B).

Fluorescence spectroscopy

The uorescence spectra of Hb/Mb were measured in thepresence and absence of CNPs. Hb shows a strong uorescencewith an emission peak at �353 nm (Fig. 2A). Mb showed asimilar uorescence behavior, however, with an emission peakat �330 nm. The CNPs showed no intrinsic uorescence in the

This journal is © The Royal Society of Chemistry 2014

Fig. 2 Effect of the compounds on the intrinsic fluorescence of Hband Mb. (A) The emission spectra of Hb (0.5 mM) as a function of CNPswith concentration varying from 0 mM to 4.5 mM (9 steps). (B) Theemission spectra of Mb (0.5 mM) as a function of CNPs withconcentration varying from 0 mM to 5.5 mM (11 steps). Emissionmaximum: 353 nm for Hb and 330 nm for Mb.

Fig. 3 Modified Stern–Volmer plot for the determination of Kd of Hbbinding with CNPs (A), and Mb binding with CNPs (B).

Fig. 4 van't Hoff plot for the determination of the thermodynamicsparameters for the binding of Hb with CNPs.

Table 1 Binding constant and the thermodynamic parameters ofbinding

Kd (mM)DG�

(kJ mol�1)DH�

(kJ mol�1)DS�

(J mol�1 K�1)

Hb-CNP 0.29 �37.98 �32.19 19.10Mb-CNP 0.25 NA NA NA

Fig. 5 Circular dichroism spectral changes of Hb (A), and Mb (B) oninteraction with increasing concentration of CNPs.

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experimental buffer solution. However, the presence of CNPs inthe solution effectively reduced the uorescence yield of Hb/Mb. For both the Hb and Mb, the uorescence intensitydecreased gradually with increasing CNPs concentration, indi-cating effective quenching of the protein uorescence. Fig. 2shows the spectra in the presence of different concentrations ofCNPs with Hb and Mb.

A temperature dependent experiment was performed toobtain the thermodynamic parameters of binding (Fig. 3). Thisexperiment showed that the quenching constant for CNPsdecreased with increasing temperature. Quenching decreasedwith increasing temperature, which suggests static quenching(Fig. 4). However, uorescence lifetimemeasurements would beadenitive indicator. Binding constants and the thermodynamic

This journal is © The Royal Society of Chemistry 2014

parameters47,48 of the binding are listed in Table 1. Here, weobserve that the entropy of the system increases whereasenthalpy decreases; therefore, the process is enthalpy driven.Weagree that the entropy change due to adsorption is negative. Inthismacromolecular system, when we consider the contributionof water exclusion/solvent release, the overall entropy of thesystem increases.49

Circular dichroism

Circular dichroism (CD) is a powerful technique to investigatethe interaction of proteins with other molecules, includingnanoparticles. In general, this technology is used to determinethe protein conformation in solution or adsorbed onto othermolecules or nanoparticles. Here the CD spectra were obtainedin the wavelength range of 200–250 nm by preparing thealiquots of the samples, the results expressed in Fig. 5.

The farultravioletCDspectraofnativeHbandMb (Fig. 5) havetwo minima at 208 (p–p*) and 223 nm (n–p*), which are char-acteristic of typical a-helical structures. The carbon nanoparticleis notCDactive. ForHbandMb, changes in theCDspectra for thesecondary and tertiary structures have been noted upon CNPbinding, but such changes are much greater for Hb compared tothat of Mb. It has been found that the binding of CNP to Hbcauses a reduction of a-helix of the protein secondary structurefrom76% (freeHb) to 61% (Hb-CNP),whereas forMba reductionof a-helix of the protein secondary structure from 75% (free Mb)to 71% (Mb-CNP) has been observed. Such results suggeststructural changes for both Hb and Mb upon binding to CNP.

Conclusion

The CNPs were synthesized and their interactions with Hb andMb were studied. The results show a remarkably strong inter-action between carbon nanoparticles with hemoglobin andmyoglobin separately. TEM, AFM and DLS experiments havebeen performed to conrm the ultrane nature of the

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synthesized CNPs. Temperature dependent uorescence spec-troscopy shows the exothermic binding of CNPs to Hb, which isfavored by enthalpy and entropy changes. Circular dichroismstudy also reects the signicant disruption of the proteinsecondary structure and partial unfolding of the helical struc-ture. These ndings have given us information regarding theunderstanding of this strong interaction between carbonnanoparticles and hemoglobin and myoglobin. This might helpto better clarify the potential risks and undesirable healthhazards of carbon nanoparticles.

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