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Research ArticleReceived: 16 January 2009 Accepted: 26 February 2009 Published online in Wiley Interscience: 8 April 2009

(www.interscience.wiley.com) DOI 10.1002/jrs.2275

Polarization Raman study of protein orderingby controllable RBC deformationSatish Rao,a Stefan Balint,a,b Luisa del Carmen Friasa,c and Dmitri Petrova,d∗

Polarized Raman spectroscopy is used to provide evidence of hemoglobin protein ordering as the red blood cell (RBC) isstretched with optical tweezers. The stretching of the cell is intended to mimic the deformation that it experiences as it travelsthrough vessels and capillaries. The depolarization ratios for a number of heme Raman bands change as the cell is stretched,confirming the semi-ordered nature of the hemoglobin ensemble in the cytoplasm. Furthermore, trends observed in the ratioshifts point to increased packing and ordering of the Hb after cell stretching. This evidence should shed more light on to therole of deformation in the RBC function. Copyright c© 2009 John Wiley & Sons, Ltd.

Keywords: polarized Raman spectroscopy; optical tweezers; red blood cell; hemoglobin; ordering

Introduction

The study of the red blood cell (RBC) by means of Ramanspectroscopy is fruitful because of its relatively simple structureand dense vibrational spectrum. Although the use of Ramanscattering can be quite complicated in biology, the spectrum forthe hemoglobin protein (Hb) is well established,[1,2] including anumber of studies of the spectral changes due to the oxygenationstate of the strongly Raman scattering heme groups.[1 – 4] For themost part, these results have pointed to a connection between thephysical conformation and chemical composition of the protein,evidenced through changes in the heme group Raman spectrum.

Recently, we completed a study that took this correspondenceone step further by establishing a connection between RBCdeformation and the oxygenation state of the Hb.[5] UsingRaman tweezers, the RBC was stretched and the Raman spectrummonitored, resulting in changes in the heme group bands thatindicated a transition from oxygenated to deoxygenated states ofthe Hb. The effect is attributed to increased rates of Hb–membraneand neighboring Hb interactions due to stretching of the cell.

Those results reiterate two factors in the oxygenation cycleof the Hb: oxygen transport across the membrane and themechanochemical changes of the Hb oxygen affinity. A thirdfactor that could be significant as well is the internal oxygentransport across the Hb’s in the cytoplasm. This should be acontributing factor because of the high Hb concentration in thecytoplasm (∼30%),[6] which naturally indicates rapid interactionsbetween neighboring proteins. The high concentration or packingof the Hb proteins in the cytoplasm has previously been thoughtto contribute to intracellular oxygen transport.[7]

The packing of a highly concentrated group of identicalstructures leads to the natural belief of the existence of orderingwith those structures. The potential role of Hb ordering in oxygendiffusion is an interesting prospect, with the first step beingto demonstrate its existence in the RBC. Early studies haveconcluded that the Hb’s are not randomly distributed as in a freeliquid;[8] rather, they reside in a semicrystalline state.[6] An orderto the packing is further supported by our recent simulation thatdemonstrated that two Hb’s in close vicinity to each other prefer toelectrostatically bind at a particular contact point.[5] Deformation

of the cell (by stretching, for example) should increase the packingof the Hb’s because of the decrease of the internal liquid volumewithout Hb loss.[9] However, whether or not this increased packingwould also force a higher ordering is still in question.

The purpose of this work is to observe the presence of Hbordering and its dependence on controllable cell deformation.Polarized Raman spectroscopy is the perfect tool for such astudy. The Raman spectrum of the Hb is consistent with highdetail, and the band polarizations can be related to the proteinordering. Raman spectroscopy alone is a fruitful technique forthis application because of the high level of information that isprovided at low cost in terms of damage to the cell. The latter is dueto the ability to use infrared wavelength excitation at low powers.However, for the most part, Raman peak positions are not affectedby the orientation of the structure. Polarized Raman spectroscopyadds this sensitivity. Studying the polarization components ofthe scattered light has long been established as a powerfulmethod to elucidate symmetries, orientations, and order/disorderin crystalline structures (for example,[10,11]). Also important, sincethe depolarization value is a ratio of intensities, the Raman intensityof each mode, which can vary from cell to cell, essentially becomesinvariant in this regime. The method has already been used tostudy Hb ordering in relaxed RBCs, with evidence of the existenceof some level of ordering in the absence of deformation.[12]

The optical tweezers adds the critical capability of dynamicallydeforming the cell in a controllable way while studying its Ramanscattering in vivo.

∗ Correspondence to: Dmitri Petrov, ICREA – Institucio Catalana de Recerca iEstudis Avancats, Barcelona 08010, Spain. E-mail: Dmitri.Petrov@icfo.es

a ICFO - Institut de Ciencies Fotoniques, Mediterranean Technology Park, Av.Canal Olimpic S/N, Castelldefels (Barcelona) 08860, Spain

b Department of Biophysics, University of Pavol Jozef Safarik, Kosice 04154,Slovak Republic

c Department of Chemistry, Universidad de Sevilla, Sevilla 41004, Spain

d ICREA – Institucio Catalana de Recerca i Estudis Avancats, Barcelona 08010,Spain

J. Raman Spectrosc. 2009, 40, 1257–1261 Copyright c© 2009 John Wiley & Sons, Ltd.

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Experimental

Sample preparation

Human serum albumin (HSA) and phosphate buffer saline (PBS,pH = 7.4) were obtained from Sigma-Aldrich. The aqueoussolutions were prepared by using deionized water. Bloodwas obtained by venipuncture from healthy volunteers andcentrifuged three times (2000 rpm, 3 min) in PBS before finaldilution 1 : 100 with HSA solution (2 mg/ml).

Raman measurement

The multitrap Raman setup employed has been describedelsewhere and used successfully for Raman studies of livingcells.[13] The cells were placed inside a custom-made sampleholder with a 80-µm-thick cover slip. The holder was then put in toan inverse Olympus IX 51 microscope equipped with a 100 × 1.3NA oil immersion objective and a micrometer-controlled x-y stage.A 785-nm beam was used for the excitation of Raman spectra withan average power of about 5 mW at the sample. The backscatteredlight was collected by the microscope objective and passedthrough a holographic notch filter and confocal system with a 100-µm pinhole. A linear polarizer was placed in front of the entranceslit of a spectrometer. The spectrometer had a 600 lines/mmgrating and was fitted with a thermoelectrically cooled (−100 ◦C)charge-coupled device (CCD). A CCD camera attached to themicroscope provided optical images during experiments. Ramanspectra were recorded with a spectral resolution of 3 cm−1. A 10%difference was found between the light intensity throughput ofthe optical beam with the polarization perpendicular and parallelto the spectrometer slit, and the corresponding corrections wereintroduced in the subsequent measurements.

A 1064-nm beam was used for the dual-spot trap. The expandedand collimated beam was directed through an interferometerbefore entering a second collimating lens system which conjugatesthe image from the movable interferometer mirror planes to theback of the microscope objective. The movement of the mirrorsin either arm of the interferometer results in the movement ofthe trap in the focal plane (x –y) of the objective without changesin its intensity and shape, thus keeping the trapping force of thetraps the same. By allowing one of the collimating lenses in frontof the interferometer to be movable along its optical axis, the

trap beams were slightly shifted axially such that they were in thesame plane as the Raman excitation beam (Fig. 1a). The beamswere passed through the same microscope objective with a totalaverage power of about 10 mW at the sample.

Figure 1b presents camera images of the equilibrium andstretched states of an RBC that is held in the dual-spot trap.The cell was aligned such that the Raman beam passed throughits center with the incident polarization directed along the cellstretching direction. Raman spectra with polarization parallel andperpendicular to the incident beam were then recorded for the cellin its equilibrium and stretched position. We define the equilibriumposition as the state where the cell is trapped by both beams suchthat it is hindered from rotation with minimal constraint in thestretch direction. The traps were positioned to keep the cell asclose to its free length as possible in order to achieve this. Forthe stretched position, the cell was stretched to the maximumextent possible by the fixed power of the 1064-nm traps, whichwas equivalent to force in the picoNewtons range and is suitablefor RBC stretching without irreversible damage.[14,15] At theseconditions, the maximum stretch length was about an additional40–50% of the original cell length on average. Only the maximumlength was tested; measurements at intermediate lengths yieldedinconsistent results. For each spectrum, a single 30-s acquisitionwas performed. To test for possible photodegradation effects,Raman spectra were recorded for RBCs held in the dual-spot trapbefore and after a time period of a few minutes. Minimal to nochanges were seen in the spectra.

Results

Figure 2 shows the full Raman spectra for the equilibrium andstretched cell for the two detected polarization directions. Thespectra represent an average of measurements from 25 cells.Adjacent point averaging was used to smooth the data and thebackground was removed using an established method.[16]

Figure 3 is a selection of bands extracted from the full Ramanspectra. The depolarization ratio (ρ) of each band was thendetermined as the ratio of the intensities of the perpendicular(I⊥) to parallel (I||) polarized Raman scattered light (ρ = I⊥/I||). Themeasured depolarization ratios before stretching were consistentfor the majority of the cells measured and agree with those from

Figure 1. (a) A rendering of the general setup of two movable 1064-nm optical traps stretching an RBC while a 785-nm beam passes through its center,exciting the Raman scattering. (b) Two camera images show the cell in its equilibrium (left) and stretched (right) position. All beams (circles) are directedin to the page (z) and the polarization direction (arrow) of the Raman excitation beam is in the plane of the cell, aligned with the direction of stretching(x). This figure is available in colour online at www.interscience.wiley.com/journal/jrs.

www.interscience.wiley.com/journal/jrs Copyright c© 2009 John Wiley & Sons, Ltd. J. Raman Spectrosc. 2009, 40, 1257–1261

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0

50(a)

Ram

an In

tens

ity/A

rbitr

. Uni

ts

800 1600

Wavenumber/cm-1

800 1600

Wavenumber/cm-1

0

60(b)

Ram

an In

tens

ity/A

rbitr

. Uni

ts

Figure 2. Full Raman spectra of the RBC in the equilibrium (a) and stretchedstate. (b) Spectra for the parallel (solid line) and perpendicular (dashed line)polarizations are given.

previous studies.[17] The depolarization ratios after stretching wereconsistent for 70% of the cells measured. However, only a few cellscontained all of the ratio changes in their entire spectrum whichnecessitated spectral averaging. In light of the presented valuesbeing averages of the cell sample set, it is important to include

uncertainties in order to have an understanding of the precision.This was taken as the standard error, defined as the standarddeviation divided by the square root of the sample size (i.e. thenumber of cells tested), which is the appropriate measure whenpresenting mean values.

Discussion

Analysis of depolarization ratios

The individual Raman bands of Fig. 3 were chosen judiciously. Theheme group bands that are known to be sensitive to the oxygena-tion level of the RBC (1250–1370 and 1600–1650 cm−1)[1,3] wereignored because the amount of oxygen in each cell studied can-not be consistent. In addition, our previous study has shown thatthe oxygenation can further change when the cell is stretched.[5]

Therefore, it was important to ignore these regions. The remainingbands are summarized in Table 1, where the depolarization ratiosare calculated from Fig. 3. Uncertainties are given in the table aspercent error and were calculated separately for each Raman banddepolarization ratio. The error varies from band to band, which isexpected because the Raman activity is not constant across a givenspectrum. We found that the limits of depolarization are unalteredafter the background subtraction process, i.e. the states of po-larization are defined as (p) polarized, ρ < 3/4, (dp) depolarized,ρ = 3/4, and (ap) anomalously polarized, ρ > 3/4.

First, the depolarization ratios of the heme Raman bands changebetween the RBC equilibrium and stretched states, which is anindication that the Hb proteins are not completely randomlymoving like in a liquid and some type of ordering in the ensembleexists.

In order to further elucidate how the band depolarizationschange, all of the measured depolarization ratios are plotted inFig. 4. All bands that are discussed in the next section have averagedepolarization ratio values that lie outside of the error range ofits counterpart in the unstretch/stretch comparison. It is evidentthat when the cell is stretched, the depolarization ratios shift awayfrom the cell equilibrium values in different ways. Four bands (670,970, 1400, and 1440 cm−1) shift toward their symmetry-dictatedpolarizations. For example, the 1400 cm−1 band is an asymmetric

0

1

156015401540 1560

stretchedequilibrium

Ram

an In

tens

ity/A

rbitr

. Uni

ts

Wavenumber/cm-1

0

1

1600158016001580

1

01460144014601440

1

01120108011201080

1

010001000 960960

1

0780750780750690660690660

1

0

1

014101410 13801380

Figure 3. Individual spectra for the extracted Raman bands. For each set, the Raman spectra for the parallel (solid line) and perpendicular (dashed line)polarizations are included for the RBC in the equilibrium (left) and stretched (right) states.

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Table 1. Summary of measured depolarization ratios for the equilib-rium and stretched states of the RBC

Mode (cm−1)ρ

Equilibriumρ

Stretched Error (%)Localcoordinate

670 0.50 0.44 9 δ(pyr deform)sym

750 0.72 0.80 8 ν(pyr breathing)

970 0.83 0.71 13 δ(pyr deform)asym

1000 0.69 0.68 10 ν(CβC1)asym

1120 0.78 0.76 5 ν(pyr half-ring)sym

1400 1.1 0.85 7 ν(pyr quarter ring)

1440 1.1 0.97 9 ν(CαCm)sym

1545 1.1 1.35 5 ν(CβCβ )

1580 1.2 0.90 13 ν(CαCm)asym

Local coordinate assignments are taken from Ref. [23].

800 1200 1600

0.45

0.90

1.35

Dep

olar

izat

ion

ratio

Wavenumber/cm-1

Figure 4. Depolarization ratio values for each band from Fig. 3 for theequilibrium (black square) and stretched (gray circle) state of the RBC.Error bars included are based on the standard error for each Raman banddepolarization mean value.

stretching mode (B2g) of the pyrrole quarter ring. The data forthis band shows a shift from anomalous toward a depolarizedstate of polarization. Conversely, the band at 670 cm−1 is a totallysymmetric in-plane bending deformation (A1g) of the pyrrole ring.When the cell is stretched, this mode becomes more polarized.

Conversely, the depolarization ratios for two bands shifttoward anomalous polarization upon cell stretching. The commonpyrrole ring stretch breathing mode at 750 cm−1 (B1g) goes fromdepolarized to anomalous polarization and a C–C stretching modeat 1545 cm−1 (B1g) that was initially anomalously polarized hasa higher depolarization ratio after stretching. A third band at1580 cm−1 (Eu) typically has a weak Raman activity; however,a strong anomalously polarized peak is measured at thiswavenumber and the depolarization ratio reduces after stretching.

Finally, the A2g type vibrations at 1000 and 1120 cm−1 shouldgive anomalous polarizations when resonantly excited in thevisible. However, both the depolarization ratios are measured tobe close to 0.75 and exhibit little change after the cell is stretchedfor our case of near-infrared excitation.

Deformation-induced heme protein packing and ordering

There are two consequences that are realized from the experi-mental data. The stretching of the RBC first packs the Hb’s more

closely together, and second, additionally, arranges the Hb’s in amore ordered fashion.

Evidence of closer packing comes from the increased depo-larization ratios of B1g vibrations after cell stretching. Anomalouspolarizations are typically possible only with resonance Ramanexcitation and with A2g type vibrations.[18] Resonance Raman isusually seen through the Soret band for hemoglobin, thus re-quiring visible wavelength excitation. However, resonance canalso be achieved at near-infrared wavelengths through a chargetransfer transition, the so-called band III absorption, which haspreviously been observed in deoxygenated Hb[19] and in an oxi-dized, toxic form of Hb.[20] In both cases, excitation at 760–780 nmcaused resonant Raman enhancement for bands at about 750and 1550 cm−1. The bands measured here at these wavenumbersindeed have strong intensities; yet their B1g type vibrations are typ-ically assumed to not allow the anomalous polarizations that havebeen measured here. However, for certain vibronic interactionsbetween the nuclear coordinate and an electronic transition, andtherefore a departure from the Franck–Condon regime, anoma-lous behavior at resonance is possible. This has been shown tooccur for B1g vibrations in porphyrin structures.[21]

Thus, the observation of anomalous polarization for thesevibrations is well supported. Conversely, the depolarization ratiosfor sensitive bands in this regime, i.e. the measured bands at750 and 1545 cm−1, can be used as markers for the level ofresonance Raman enhancement. Indeed, the depolarization ratioof anomalously polarized bands have been shown to vary onthe basis of the resonance Raman excitation wavelength[22]

even for B1g type vibrations.[21] Aside from wavelength tuning,enhancements can also be a result of closer packing of theHb’s because of the increase in excitonic interactions betweenneighboring heme groups. The increased coupling does notrequire direct covalent linking, because it could occur throughvan der Waals contacts or simply through the delocalized space,both of which are more likely to be probed at longer wavelengths.This effect was observed in β-hematin, which is a system ofpacked or aggregated porphyrins.[20] Hence, the RBC stretchingpacks the Hb’s more, which is indicated by the increase of thedepolarization ratios of the two bands that exhibit resonant Ramanand anomalous polarization behavior at near-infrared excitation.

The closer packing of identical structures leads to the idea of or-dering. Previously, we conducted a simulation that demonstratedthat when two Hb proteins are brought within close proximity, themost favorable configuration is achieved when there is contactat the F-helix.[5] Thus, forcing closer packing of these proteinsshould cause them to reorient themselves in an ordered fashion.There are differences in the depolarization ratios of some hemeRaman bands when comparing spectra from the RBC,[12] where thehemoglobin is in a semicrystalline state in the dense cytoplasm,to those from Hb solution.[17] If the proteins are moving toward afixed array, then band depolarizations that are not affected by theband III near-infrared resonant excitation should migrate towardtheir symmetry-dictated values. That is the case here, evidencedthrough the shifting of depolarization ratios of three B2g vibra-tions toward 0.75 and one A1g vibration toward 0 after the RBC isstretched.

Although the observed cell deformation-induced effect iscompelling, the result should certainly be dependent on thesymmetry of the protein packing which determines the effect ofthe incident beam polarization–sample orientation. The symmetryof the Hb ordering is difficult to determine from our present data.One way to test this would be to vary the cell–incident beam

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polarization orientation. As mentioned previously, a study such asthis has already been done for two cell orientations, in the absenceof stretching, at fixed incident polarization.[12] In our case, simplyrotating the incident polarization by 90◦ would be incomplete.Expanding upon this requires a complete measurement over allpossible configurations, i.e. for incident polarizations in and normalto the plane of the cell. This could give a complete picture of theHb packing. A test such as this is not possible in the current setupbecause the RBC always settles in to the optical traps with its planeparallel to the beams, allowing the cell to refract the trapping lightthe most, thus maximizing its stability. Our current efforts involveattaching micro-sized handles to the cells in order to allow a fullerrange of manipulation.

Conclusion

The effect of RBC deformation on Hb ordering was studied.Polarized Raman scattering components were collected for theequilibrium and stretched states of the cell. Consequently, it wasshown that upon stretching, the depolarization ratios for a numberof Hb heme Raman bands change. The shifting of the values is itselfan indication of the existence of Hb ordering. The characteristics ofthe movements of some of the band depolarization ratios indicatethat the Hb’s are being more packed as the cell is stretched, whilethose of other bands indicate that the protein ensemble is beingpushed to a more ordered state. In order to further advance theunderstanding of the packing configuration, additional testing ofthe dependence on cell–incident beam polarization orientations isrequired, which is a motivation for future work. This phenomenonis another example of how RBC deformation plays a larger rolein the cell’s function, this time through manipulation of the Hbensemble.

Acknowledgements

We acknowledge support from MEC FIS2005-02129 and FundacioCellex Barcelona.

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J. Raman Spectrosc. 2009, 40, 1257–1261 Copyright c© 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/jrs