pH Induced Structural Modulation and Interfacial Activity of Hemoglobin at the Air/WaterInterface

Mrityunjoy Mahato,† Prabir Pal,† Tapanendu Kamilya,†,‡ Ratan Sarkar,† andG. B. Talapatra*,†

Department of Spectroscopy, Indian Association for the CultiVation of Science, JadaVpur, Kolkata-700032, India,and Department of Physics, Narajole Raj College, Narajole, Paschim Medinipur-721211, India

ReceiVed: August 21, 2009; ReVised Manuscript ReceiVed: NoVember 13, 2009

In this Article, we report the surface activity of the human globular blood protein, hemoglobin (Hb), at theair/water interface. The Langmuir-Blodgett technique is used for monolayer characterization. The adsorptiongrowth-kinetics study shows that the adsorption process at the air/water interface is involved with twomechanisms: one diffusion with adsorption and the other rearrangement with unfolding. The kinetics is foundto be dependent on pH and protein concentration in the subphase. The CD and FTIR studies suggest largerintermolecular aggregate and -sheet formation in the film lifted from the air/acidic water subphase. In alkalinepH and in isoelectric pH (6.8), not much variation is observed. The FE-SEM images support this observation.The acidic pH induced such conformational changes, and aggregation is explained with the argument ofR-helix to -sheet conversion as well as the competition between protonation and deprotonation of the aromatic-amino acid residues at the air/water interface.

1. Introduction

The study of proteins/enzymes at the molecular and/orsubmolecular level is a subject that has received considerableattention in the development of both fundamental and potentialapplications in various scientific fields, such as nanobioscience,material science, and molecular biology/biotechnology.1-6 Theimmobilization of proteins/enzymes onto an appropriate surfacewith minimal denaturation is extremely important for theirbiological activity and application in biomolecular devices.7-9

Recently, a variety of methods has been developed to im-mobilize biomaterials onto solid surfaces, such as the sol-gelprocess, layer-by-layer, self-assembly, Langmuir-Blodgett (LB)techniques, etc.10-19 Among these methods, the LB techniqueis the most versatile and efficient technique for protein/enzymeimmobilization to obtain good molecular organization/architec-ture with control at the molecular level.20-22 In the immobiliza-tion process of protein/enzyme onto a suitable substrate, thedenaturation, unfolding, and aggregation are the key issues asfar as the biological activity is concerned.23-25 On the other hand,the fundamental understanding of the molecular mechanismrelated to the protein aggregation, unfolding, is also anotherimportant issue, as it involves the pathology of many kinds ofdiseases such as neurodegenerative diseases, Alzheimer’sdisease, etc.25-28

In this Article, the interfacial activity, reorganization, unfold-ing, intermolecular aggregation, and conformational regulationof Hb at the air/water interface is studied as a function of pHand the concentration of Hb (CHb) of the subphase, by monitor-ing the surface activity of Hb from adsorption growth kinetics(π-t), pressure-area (π-A) isotherm, and compressibility(-π) studies. Circular dichroism (CD) and Fourier transforminfrared (FTIR) spectroscopy were employed to characterize the

states of conformation as well as conformational change of Hbin the films lifted from subphases having different pH’s. Inaddition, the pH dependent structural and morphological aspectsof the transferred LB films of Hb under different conditionswere characterized by high-resolution field emission scanningelectron microscopy (FE-SEM).

2. Experimental Section

2.1. Materials. The Human Normal Hemoglobin, lyophilizedand stored at 2-8 °C, was purchased from Sigma ChemicalCo. and was used as received without further purification. HCland NaOH were obtained from Merck India Ltd.

2.2. Methods. 2.2.A. Adsorption Growth Kinetics (π-t) atthe Air/Water Interface. To study the (π-t) adsorption growthkinetics at air/water (pH controlled) interfaces, a fixed volume(10 mL) of aqueous solution of Hb of a particular concentrationwas injected into the subphase of volume 750 mL to attain therequired final concentrations of Hb in the subphase of the LBtrough. The final CHb values used in our study are 0.0001, 0.001,0.002, 0.006, 0.008, and 0.01 mg/mL. The pH was adjusted byadding HCl and NaOH into the pure water subphase. The pHadjustment was done prior to protein addition.

The computerized LB trough used was of the Teflon-bar-barrier type (model 2000C, Apex Instruments Co. India)enclosed in a plexiglass box to reduce film contamination andequipped with a Wilhelmy type balance, to an accuracy of (0.01mN/m. The trough width and length are 200 mm and 450 mm,respectively. This work was performed at constant area (200mm × 100 mm) of the LB trough by placing the barrier at theappropriate position. The depth was about 40 mm. Tripledistilled and deionized water, purified using a Milli-Q Apparatus(Millipore, USA), was employed to prepare the subphase andthe protein solution. The pH and the resistivity of the freshlyprepared water were 6.8 and 18.2 MΩ cm, respectively. Allexperiments were performed at a temperature of 25 ( 0.5 °Cunless otherwise mentioned. At least three independent runswere performed to check the reproducibility.

* To whom correspondence should be addressed. Phone: +91-33-24734971. Fax: +91-33-24732805. E-mail: spgbt@iacs.res.in.

† Indian Association for the Cultivation of Science.‡ Narajole Raj College.

J. Phys. Chem. B 2010, 114, 495–502 495

10.1021/jp908081r 2010 American Chemical SocietyPublished on Web 12/07/2009

2.2.B. Pressure-Area (π-A) Isotherm Measurements. Forpressure-area (π-A) isotherm measurements of Hb on the pHcontrolled water subphase, the aqueous solution of Hb havinga concentration of 0.1 mg/mL was spread on the water subphase.The spreading was done using a microsyringe in such a mannerthat the surface pressure would not rise above 0.5 mN/m. Thenumber of molecules thus spread was calculated using themolecular weight of Hb monomer as 17 kDa.29 After 20 min ofwaiting time, the monolayer was slowly compressed with acompression speed of 1 Å2/(molecule-min).

2.2.C. Monolayer Transfer. The monolayers were transferredvery carefully through up stroke with a speed of 5 mm/minand at a constant surface pressure onto hydrophilic glasscoverslips, silicon wafers, and quartz slides, which werepreviously immersed in the subphase. The details of theprocedure of cleaning of the substrates were described else-where.30

2.2.D. Study of Surface Morphology. A high-resolution fieldemission scanning electron microscope (FE-SEM, model no.JEOL JSM-6700 F) with a range of 0.5-30 kV with a lateralresolution in the range 1.2-2.2 nm was employed to measurethe surface morphology of all transferred LB films on the glasssubstrate.

2.2.E. Measurements of FTIR Spectra. FTIR spectra of LBfilms lifted at 20 mN/m from pH controlled subphases at pH 3,6.8, and 10 on silicon wafers were recorded at room temperatureon a Magna-IR instrument (model no. 750 spectrometer, seriesII), Nicollet, USA. In all cases, the data were averaged over100 scans. The resolution of the instrument was 4 cm-1.

2.2.F. Measurements of CD Spectra. CD spectra of LB filmslifted at 20 mN/m from pH controlled subphases at pH 3, 6.8,and 10 were recorded at room temperature on a JASCO J-815CD spectrometer (model no. J-815-150 S). For solution, we usea 1 mm path length cuvette. Three scans were adapted todecrease the noise of the CD spectra.

3. Results and Discussion

3.1. Characteristics of π-A Isotherms. Figure 1A displaysthe pressure-area isotherm of Hb monolayer on the surface ofsubphases at different pH’s. The surface pressure starts toincrease due to isothermal compression. From Figure 1A, themean area/molecule values, estimated by extrapolating thesteeply rising part of the π-A isotherms to π ) 0, are ∼8, 22,and 13 nm2/molecule at pH 3, 6.8, and 10, respectively.However, this type of estimation for a less steep isotherm maynot be appropriate. According to Helm et al.,31 it will be morereliable to estimate the area/molecule at a particular pressurebefore the collapse region of isotherm. We have estimated thearea/molecule at 20 mN/m pressure as 5, 16, and 7 nm2/moleculeat corresponding pH’s of 3, 6.8, and 10, respectively. A bardiagram of area/molecule at 20 mN/m surface pressure withdifferent pH’s of the subphase is shown in the inset of Figure1A. Our feeling is that the probable value should be in betweenpressure onset and collapse. In this context, it is necessary tomention that, according to the Protein Data Bank (PDB),32 theestimated maximum diameter of an individual monomer of Hbis ∼4.5 nm. Hence, the area/molecule value of a Hb monolayershould be around 20 nm2. The inset of Figure 1B shows thestructure and dimension of Hb monomer obtained from thePDB.32 It is to be noted that the obtained area/molecule fromthe π-A isotherm for the subphase at pH 6.8 is very closeto the estimated value from the PDB. However, at pH 3 and10, the obtained area/molecule values are less than the estimatedvalues. This may arise not only due to the solubility of the Hbmolecules in the water subphase for which they may sink or

dissolve into the water subphase but also due to the fact thatthe orientation of the molecules is occupying a smaller area atthe surface.33-36 It is interesting to note that the measured area/molecule values on either side of the isoelectric point (pI) ofHb (6.86)37 are less. Here, changes in surface pressure of theHb monolayer on aqueous subphases during isothermal com-pression are supposed to be only due to the structural rear-rangement of hydrophobic moieties of Hb molecules alignedtoward the air at the air/water interface and there are severalpieces of literature on this topic.30,38-41

The π-A isotherm of Hb shows a gaseous (G) state betweensurface pressures of 0 and 2 mN/m, where the area/moleculedrops from 40 to 23.5 nm2. It is obvious from Figure 1A thatmore compression makes π increase monotonously up to anupper limit of π ∼22 mN/m on the pure water subphase, wherethe monolayer is in the liquid (L) state of Hb. This kind of Lstate was observed in previous literature, in the case of proteins/enzymes like ovalbumin, apolipoproteins, and alcohol dehydro-genage.25,30,42 Further compression of the system between π )25 and 35 mN/m shows a slight bending toward the left forisoelectric pH. Careful observation shows that this bending isnot prominent for acidic or for alkaline subphases. This bendingof the isotherm toward lower area/molecule may account forsome structural transformation at the restricted condition of themonolayer.25,30 Here, the changes of the transition region atdifferent pH’s associated with the isotherms are prominent fromthe compressibility study, which is discussed in the next section.

Figure 1. Panel A represents π-Α isotherms at room temperature(26 °C) of Hb containing subphases at different pH’s. The inset in panelA is the plot of area/molecule (measured at 20 mN/m) at different pH’sof the subphase. Panel B represents a -π plot at different pH’s ofthe subphase obtained from π-Α isotherms. The inset in panel Brepresents the monomer structure of Hb collected from the Protein DataBank. The line indicates the approximate width, which is found to beabout 4.5 nm.

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3.2. Compressibility (-π) Analysis. It is seen from Figure1A that there is a distinct difference in nature among theisotherms, at acidic pH, isoelectric pH, and alkaline pH. Thischanges mainly around 25-35 mN/m. To visualize the transition(slope change) more prominently, we have performed compress-ibility (-π) analysis. This is an important tool to characterizethe transition regions. Here, is represented by the followingequation.25,43

) -(1/A)(δA/δπ)T (1)

The appearance of peaks in the -π curve can represent sometransitions, and the peak heights indicate the maximum com-pressibility (max) of the monolayer. The asymmetry of the peakimplies that the transition may consist of several steps.43 The-π curve (Figure 1B) obtained for the subphase at pH 6.8shows a broad asymmetric peak at a higher region of π, between30 and 40 mN/m, with max ) 0.098 m/mN at π ) 34 mN/m.This may be due to a partial squeeze-out of the protein fromthe monolayer preceding the collapse.37 On the other hand, forthe alkaline subphase (pH 10), the transition is observed with amuch lower compressibility (max ) 0.021 m/mN at π ∼32 mN/m), and for the acidic subphase (pH 3), no such peak is observed.The absence of any slope change at acidic pH may be due tothe formation of some aggregate before the transition occurs.

In the case of proteins/enzymes kept at different pH’s, thewater molecules inside the cavity of the protein molecule aredifferent from bulk water molecules and play an important rolein the process of folding and overall stability of the proteinmaking hydrogen bonds among the polar groups of the aminoacid residues.44-47 In our system, the monolayer of Hb at acidicpH may be favorable for hydrogen bonding between the polargroups of the amino acid residues.

3.3. Study of Hb Adsorption at the Air/Water Interface.3.3.A. Adsorption of Hemoglobin at the Air/Water Interface.To explore the surface activity of protein, we have performedthe adsorption growth kinetics (π-t) of Hb. Several featuresare seen in the π-t adsorption curves of Hb. In Figure 2A, wehave plotted the variation of surface pressure with time at thepure water subphase (pH 6.8) with varying concentration of Hbinjected into the subphase. Hb is surface active and adsorbs atthe air/water interface from the subphase.

We observed that, at a low concentration of Hb, growth of πstarts after an initial lag time (τlag), where π values remain nearzero. Moreover, τlag decreases with increasing concentration.The initial lag phase is the significant characteristic of the proteinadsorption process. In this region, the interface possesses aninsufficient amount of Hb molecules for a noticeable change inπ. Due to the scarcity of Hb molecules at the air/water interface,on average, they are far apart, as they are in the gaseous phase.Hence, there is very little or no interaction between neighboringHb molecules. In the course of time, the number of Hbmolecules at the interface increases and they eventually comecloser to each other within their interaction radius. As a result,the surface pressure starts increasing after the period of τlag. Asthe interface becomes populated, the surface pressure increasesslowly with time and approaches a constant value (Figure 2A).

This is a typical observation of diffusion limited docking ofsurface-active protein at the interface.33 Ybert et al.35 describedthis initial lag phase as a diffusion-controlled regime andinfluence of free convection. τlag can thus be defined as the timerequired for attaining the minimum monolayer coverage for aneffective and measurable surface pressure.35 Matthew andAbraham48 describe the protein adsorption at the air/waterinterface as depending on the protein-protein and protein-surfaceinteraction strength.

When CHb is very small (0.0001 mg/mL), the surface pressurealmost saturates at π ∼1 mN/m within 1000 s. Further increaseof CHb makes the adsorption faster, and less time is needed forsaturation (Figure 2A).

The nature of the variation of τlag with CHb presented in theinset of Figure 2B shows that, with the increase of CHb, τlag

decreases. Similar observations were also reported earlier in thecase of pepsin and bovine serum albumin (BSA).34,49

3.3.B. Adsorption Growth Kinetics (π-t) of Hb at the Air/Water Interface. The π-t adsorption growth kinetics of proteingenerally follows single and/or double exponential associationkinetics using the following equations.34,49

πt ) π0 + A1[1 - exp(-t/t1)] (2)

πt ) π0 + A1[1 - exp(-t/t1)] + A2[1 - exp(-t/t2)] (3)

where πt and π0 in eqs 2 and 3 are the surface pressures attimes t ) t and t ) 0, respectively. Constants A1 and A2 are therelative contributions, and t1 and t2 are the time constants ofthe two mechanisms (diffusion with adsorption and rearrange-ment with unfolding) involved in the adsorption process.34

Though the interpretation of the parameters A1 and A2 is notvery straightforward, a relative comparison of both ofthemechanisms may be obtained from these parameters.

Figure 2. Panel A: the adsorption kinetics of Hb at the air/waterinterface. Here, the curves a, b, c, d, e, and f represent the variation ofsurface pressure with CHb ) 0.0001, 0.001, 0.002, 0.006, 0.008, and0.01 mg/mL, respectively. Panel B: the same curves from panel A aftersubtraction of the initial part (the lag time). The curved lines are thefitting curves using eq 2 for curve a and eq 3 for curves b-f,respectively. The inset is a plot of lag time tlag with CHb, which showsthat, with the increase of CHb, tlag decreases. The solid lines are thespline fit.

Hemoglobin at the Air/Water Interface J. Phys. Chem. B, Vol. 114, No. 1, 2010 497

Figure 2B shows the growth curves after discarding the initialpart belonging to the lag time and rescaling the adsorptioncurves, where the zero time is adjusted when the pressure startsto increase. The growth curves show reasonably good fits withresidual square correlation coefficient (R2) g 0.99. The para-meters resulting from the fits are summarized in Table 1.

We have tried to fit both of the fitting equations to theexperimental data. Only at very low CHb (0.0001 mg/mL), thedata is truly a single exponential one-step process. At this verylow concentration, a negligible number of Hb reaches the surfaceand hence the change in surface pressure by their unfoldingand reorganization becomes negligible. Only the process ofdiffusion with adsorption contributes to the kinetics. On the otherhand, at higher CHb (0.001-0.01 mg/mL), the double exponen-tial adsorption mechanism gives an acceptable fit rather thanthe single exponential mechanism.

With increasing CHb, the value of t1 decreases and A1

increases, implying that the diffusion of Hb is becoming fasterwith increasing CHb and uptakes of more Hb occur. In addition,decrease of t2 with increasing CHb suggests that the reorganiza-tion of Hb at the air/water interface is faster with less unfolding(decreasing value of A2) of protein. At higher CHb, the air/waterinterface is already covered with Hb molecules.

We have also studied the effect of pH on solubility andsurface activity of the protein. Experiments are performed atdifferent subphase pH’s like 3.0, 5.0, 6.8, 9.0, and 10.0. Figure3 displays the behavior of the adsorption of Hb with a fixedHb concentration of 0.002 mg/mL, in various pH’s of the watersubphase. A clear difference in the adsorption and saturationbehavior at different pH’s is observed.

In general, the variation in solubility of the protein/enzymein the bulk may occur in the presence of an electrolyte solutionand it has been reported that solubility increases on either sideof the isoelectric point of the protein.34,50,51 When an acid or abase is introduced to change the pH of the subphase containingprotein, several interactions between different entities such aswater, ion, and protein may occur and determine finally whetherthe protein is a macrocation or a macroanion.46 Thus, closest topI, the minimum solubility occurs due to the almost zero netcharge of peptides. According to the site dissociation model,34,52

the surface charge of protein arises due to protonation at acidicpH and deprotonation at alkaline of the surface functional groupsfor different pH controlled media. The amount of net chargesincreases as the pH moves away from the pI and ionic moietiesare known to be responsible for solubility. Therefore, theenhanced maximum surface activity of Hb is found at pH 6.8,i.e., near the isoelectric point, and lesser surface activity on eitherside of pI is expected. Therefore, the interfacial dynamics ofHb is larger at pH’s closest to the pI and lesser at pH’s furtherfrom the pI.

The inset of Figure 3A shows a pH vs lag time plot. It isfound that the lag period is minimum at isoelectric pH andincreases toward acidic or alkaline pH. This indicates that thesurface activity of Hb becomes less in acidic as well as in

alkaline pH. At isoelectric pH, the adsorption and saturation ofHb is fastest, having a minimum lag time. After the lag period,π rises sharply and reaches a saturation value (∼29 mN/m).Since the pI of Hb is 6.86,36 the observed faster growth of π atpH 6.8 is due to insolubility of Hb in water. Thus, at pH 6.8,more Hb molecules come to the surface very quickly and thusthe π-t curve becomes stiffer.

The adsorption kinetics is fitted by a double exponentialequation, and the fitted parameters are presented in Table 2.The initial component t1 is found to be highly pH dependent.At pH 6.8, closest to the pI, the value of t1 is much less and A1

is relatively higher than the other subphases, indicating that, inthis case, the diffusion process becomes faster with a largercontribution. A very small value of t2 implies that the processof rearrangement with unfolding is very fast. The observationis consistent with our earlier argument that at isoelectric pHthe surface coverage rate is faster. The values of t2 for pH 3

TABLE 1: Fitting Parameters of the (π-t) Adsorption Growth Kinetics of Hb with Different CHb by Single and DoubleExponential Equations

CHb (mg/mL) A1 t1 (s) A2 t2 (s) R2

0.0001 2.08 ( 0.001 818.83 ( 4.0 0.9490.001 9.00 ( 0.002 567.88 ( 4.9 23.84 ( 0.03 2466 ( 4 0.9980.002 15.00 ( 0.06 510.62 ( 5.1 16.19 ( 0.05 2110 ( 15 0.9980.006 16.06 ( 0.07 439.98 ( 9.8 16.00 ( 0.09 2092 ( 2 0.9990.008 19.16 ( 0.05 39.35 ( 0.69 11.41 ( 0.04 1362 ( 25 0.9920.01 27.75 ( 0.08 24.38 ( 0.24 1.389 ( 0.08 395 ( 32 0.943

Figure 3. Panel A: pH dependent adsorption kinetics of Hb with CHb

) 0.002 mg/mL at different pH values of 3.00, 5.00, 6.80, 9.00, and10.00 of the subphase. The inset shows the variation of lag time atdifferent pH’s (the solid line is the spline fit). Panel B: the same curvesfrom panel A after subtraction of the initial part (the lag time). Thecurved lines are the fitting curves from the double exponentialassociation equation, eq 2.

498 J. Phys. Chem. B, Vol. 114, No. 1, 2010 Mahato et al.

and 10 are higher; especially in the case of acidic pH, t2 isincreased drastically. Due to the low surface activity in acidicpH, the surface coverage rate is found to be less. Structuralreorganization through aggregate formation also may take placeslowly.

3.4. Surface Morphology of the Transferred LB Mono-layer. The surface morphologies (FE-SEM images) of thetransferred LB monolayer of Hb at pH 3.00 and 10.00 lifted at20 mN/m are presented in Figure 4. Figure 4A shows the FE-SEM images (with a magnification of 30 000) of the LBmonolayer of Hb at a subphase pH of 3.00, demonstrating thatthe film consists of aggregated structures of Hb of sizes ∼60 to200 nm. On the other hand, Figure 4B (with the samemagnification as panel A) shows the FE-SEM images of thefilms at pH 10.00, representing a homogeneous monolayer ofHb having a size of each globular unit of ∼25-30 nm. Atisoelectric pH (6.8), we have found a very similar structure(figure not shown) as that obtained at pH 10. Since the estimatedmaximum diameter of an individual monomer of Hb from the

PDB is ∼4.5 nm,32 one can roughly estimate the average numberof monomers in each globular unit of Hb within the LB filmlifted at pH 10.00 is ∼5-6 depending on the orientation of themolecule in the restricted geometry in LB film. It is known thattertiary structure of Hb in an aqueous medium is tetramer innature.53 Though unfolding of protein is likely in air, ourestimation at pH 10 and at isoelectric pH, the observed domainsin FE-SEM, may be the structure of tetramer or higher tertiaryaggregates. However, at acidic pH, higher aggregates areformed. The FE-SEM image supports the observations and thearguments of the -π compressibility as well as π-t adsorptiongrowth kinetics.

3.5. FTIR Study. FTIR spectra of LB film of proteins/enzymes are very a useful tool to identify the unfolding,denaturation, and intra- and intermolecular aggregation ofproteins/enzymes by monitoring their amide bands within a fixedrange.25,54,55 The amide band of protein mainly is comprised ofamide-I (∼1600-1700 cm-1) and amide-II (∼1500-1600cm-1). The amide-I band resulted from carbonyl stretchingvibrations of the peptide backbone, which depends on thestrength of the hydrogen bond and the interactions between theamide units; also, amide-II is due to the combination of N-Hin-plane bending and C-N stretching vibrations of peptidegroups.56,57 The vibration band of amide-I is thus sensitive tothe peptide secondary structures that span different components,such as R-helix, -sheet, turns, unordered structures, or randomcoil, and intra- and intermolecular aggregates.56,58 We haveanalyzed the amide-I band to find out about the intra- andintermolecular aggregates as well as the formation of -sheetsat different pH’s in a monolayer of Hb. However, the mostcrucial point is the assignment as well as the deconvolution ofthe different components of secondary structure in the amide-Iband. This is, generally, done by Gaussian fitting to describethe components.25,59,60 A multiple peak fitting technique withGaussian profile was employed to fit the normalized amide-Iband which allows one to identify and determine the peakfrequencies of different components. In addition, the percentagearea of the corresponding deconvoluted peaks gives the relativeamount of components. Here, the maximum number of com-ponents (N), which can be identified in the deconvoluted amide-Iband, is 5 for a meaningful fitting. Also, for a lesser/greaternumber of peaks, the fitting improve neither R2 values nordefinite peak positions.

Furthermore, the secondary structures are stabilized byhydrogen bonds between amide CdO and NsH groups; theposition of the components depends on the patterns and thestrength of the hydrogen bonds. In support of stronger hydrogenbonding, the vibration is observed at higher wavenumber. Thus,-sheet structures have the strongest hydrogen bonds, exhibitingan amide-I maximum at much lower frequency than R-helices.57-61

In the case of inter- (A1 component) and intramolecular (A2

component) aggregates, the hydrogen bonds formed betweenCdO and NsH groups of any polypeptide strands with which

TABLE 2: Fitting Parameters of the (π-t) Adsorption Kinetics of Hb (CHb ) 0.002 mg/mL) Using the Double ExponentialAssociation Equationa

(pH)Subphase A1 t1 (s) A2 t2 (s) R2

3.00 0.80 ( 0.0.001 3200 ( 1.5 55.00 ( 0.06 37 825 ( 11 0.9705.00 8.00 ( 0.07 1385 ( 4.6 38.5 ( 0.04 15 382 ( 10 0.9976.80 15.00 ( 0.06 510 ( 5.1 16.19 ( 0.05 2110 ( 15 0.9989.00 14.5 ( 0.05 754 ( 3.9 11.34 ( 0.05 3009 ( 13 0.99810.00 4.00 ( 0.06 1700 ( 5.1 16.40 ( 0.05 8234 ( 7 0.997

a A1 and A2 are the relative amplitudes, and t1 and t2 are the corresponding time constants of two mechanisms involved in the associationprocess. R2 is the residual square correlation coefficient.

Figure 4. FE-SEM images of LB films of Hb lifted at π ) 20 mN/m.Panel A: at pH 3.00 with a 100 nm scale bar and magnification of30 000. Panel B: at pH 10.00 with a 100 nm scale bar and magnificationof 30 000. The insets in panels A and B are at a higher magnificationof 100 000 and have a 100 nm scale bar (white bar).

Hemoglobin at the Air/Water Interface J. Phys. Chem. B, Vol. 114, No. 1, 2010 499

they come in contact. Thus, in consequence, many hydrogenbonds are formed between polypeptide chains in neighboringprotein molecules, forming an aggregate, and are stabilized byvery strong intermolecular hydrogen bonds.

In Figure 5, panels A, C, and E show FTIR spectra of a LBfilm of Hb lifted at 20 mN/m at pH 3.00, 10.00, and 6.8,respectively. In addition, panels B, D, and F represent theircorresponding deconvoluted amide-I spectra. An uncorrectedunsmoothed FTIR spectrum in the range 1000-2000 cm-1 fora Hb monolayer on a silicon wafer is available as SupportingInformation. A monolayer is placed on both sides. Thus,actually, we are measuring the IR for a bilayer. The percentageof secondary structural conformations is shown in Table 3 and

in a bar diagram at either pH in Figure 6A. We have also plotted-sheet over R-helix and inter- over intramolecular aggregatesto visualize more clearly in panels B and C of Figure 6. OurFTIR data indicate that the formation of -sheet and intermo-lecular aggregate is larger at an acidic pH of 3. Thus, we canconclude about the reason behind such kind of formation of-sheet and intermolecular aggregate is due to the hydrogenbonds between polypeptide chains in neighboring proteinmolecules. Protonation may occur when we make the acidic(pH 3) subphase and inject the Hb molecules that are slowlyadsorbed at the interface and getting unfolded. This may favorthe formation of larger amount of hydrogen bonding in betweenthe polypeptide chains of neighboring unfolded Hb molecules.

Figure 5. pH dependent FTIR spectra of LB films of Hb lifted at π ) 20 mN/m. Panels A, C, and E are the amide bands of Hb at pH 3, 10, and6.8, respectively. Panels B, D, and F are the deconvoluted normalized amide-I band of Hb at pH 3, 10, and 6.8, respectively.

TABLE 3: Fitting Parameters Using Multipeak Fitting of the Amide-I Band for Hb LB Filma

area (%) position (cm-1) fwhm (cm-1)

conformer A B C A B C A B C

A1 05.65 04.84 4.50 1625.0 1621.5 1622.1 13.24 13.38 13.31 16.97 42.07 25.00 1633.6 1642.2 1636.7 23.23 21.15 18.68R 43.30 43.80 47.70 1651.5 1658.0 1651.7 22.60 19.47 15.10T/Coil 22.48 08.95 11.10 1661.5 1650.3 1663.4 18.08 13.02 15.19A2 11.60 00.34 11.70 1673.8 1676.7 1677.9 14.20 12.18 14.41

a A, B, and C represent LB film lifted at pH 10, 3.0, and 6.8, respectively. Area (%) ) 100 represents the total area under the curve. fwhm) full width at half-maximum of a peak.

500 J. Phys. Chem. B, Vol. 114, No. 1, 2010 Mahato et al.

The results support our earlier observation through -π, π-t,as well as FE-SEM study.

3.6. CD Spectroscopy. We have also studied CD spectros-copy of a LB film of Hb. The far UV-CD spectra of Hb in LBfilm lifted at 20 mN/m from different pH subphases are shownin Figure 7. It is necessary to admit that, in film, sample CDspectra always include the contribution of linear dichroism(LD).62 However, as we are studying with globular protein, thiscontribution would be minimal. This is used to study the changein pH induced interfacial aggregation of Hb by monitoring thepeaks of the corresponding R-helix and -sheet. It is knownthat, when a polypeptide chain in almost all proteins existed inthe R-helical conformation, two negative bands at ∼209 and∼222 nm and a positive band at ∼190 nm are observed.43,63,64

The 209 nm band corresponds to the π-π* transition of R-helix,whereas the 222 nm band corresponds to π-π* transition forboth R-helix and random coil.61 On the other hand, for theexistence of a -sheet, a positive and a negative band appearedat 190 and 215 nm, respectively.65,66

The CD spectrum of Hb has broad negative ellipticity at 209and 223 nm together with a strong positive maximum at ∼193nm. These are the characteristics of R-helical conformation. Theintensity of the three CD bands reflects the amount of helicityin the protein. Since the structure of Hb is predominatelyR-helical, we measured the CD spectra of different LB filmsformed in different pH subphases to find out the secondary

structure67 and tried to estimate the extent of pH inducedconformational change of Hb in LB film. In addition, similarliterature is there for pH induced -rich structure in the case ofa naturally occurring enzyme, namely, organo phosphorushydrolase (OPH).66 The intensity change of these absorptionpeaks clearly shows the change of secondary structure to someextent. Our CD data show that, at pH 6.8, it is mostly in theR-helical conformation and at pH 10.00 its amount of helicitydecreased a bit but the R-helical identity remains intact. On theother hand, at pH 3.00, R-helix is lost to some extent andformation of -sheet is clearly indicated through the smallnegative peak around 215 nm. The CD spectroscopy observationcorroborates with the earlier methods.

4. Conclusion

In this work, the interfacial activity, unfolding, and reorga-nization of Hb at the air/water interface as a function of subphasepH are studied by monitoring in situ π-A isotherms, -πcompressibility, and π-t adsorption growth kinetics. Theadsorption growth kinetics study shows that the adsorptionprocess at the air/water interface involves two mechanisms: onediffusion with adsorption and the other rearrangement withunfolding. The intermolecular aggregates and -sheet formationalong with the unfolding of Hb at the interface are studied byCD and FTIR spectroscopy. The CD and FTIR studies suggesta larger intermolecular aggregate and -sheet formation in thefilm lifted from the air/acidic water subphase. The FE-SEMimages support this observation. On the other hand, at alkalineas well as at isoelectric pH, it forms a smaller uniform tertiarytetramer structure of the LB film.

Acknowledgment. We thank DST, Government of India(Project No. SR/S2/CMP-0051/2006), for partial financialsupport. M.M. thanks CSIR, Government of India, for providingthe CSIR-NET fellowship.

Supporting Information Available: The uncorrected un-smoothed FTIR spectrum in the range of 1000-2000 cm-1 fora Hb monolayer on a silicon wafer. The monolayer is placedon both sides, and hence, it is actually a bilayer. This materialis available free of charge via the Internet at http://pubs.acs.org.

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