VOL. 25 NO.

5 (2013)

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Highlighting the importance of utilising the polarisation properties of resonance Raman scattering in obtaining molecular information
Kit D. Jernshj and Sren Hassing Department of Chemical Engineering, Biotechnology and Environmental Technology, Faculty of Engineering, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark. E-mail: kdj@kbm.sdu.dk

Introduction
A Raman spectrum reflects the vibrational motion of the nuclei within a molecule. When the wavenumber of the excitation laser is chosen to be close to that of an electronic absorption of a chromophore in a biomolecule, a resonance Raman spectrum is produced. Local molecular information is obtained since only those vibrations associated with the electronic transition in the chromophore are enhanced in the resonance Raman spectrum. The polarisation of the inelastically scattered light may be different from that of the incident laser light, the difference being determined by the nature of the nuclear vibrational motion. In resonance Raman scattering (RRS), the amount of structural and chemical information deduced can be increased by analysing the polarisation of the inelastically scattered light. The polarisation is defined by the depolarisation ratio (DPR), i.e. the ratio between the scattered intensity with perpendicular polarisation compared to the incoming light and that which is parallel polarised. Since the Raman process is described by a tensor (the polarisability), it is possible to gain additional molecular information from polarised measurements on randomly oriented systems such as solutions.1,2 The benefits of analysing the polarisation effects should be considered in www.spectroscopyeurope.com

contrast to using only the unpolarised resonance Raman spectra in a classification of molecular species, detecting an aggregation or conformational change. The decision is in the latter case (using only the unpolarised spectrum) based on either occasional small wavenumber shifts of the Raman modes and/or relative intensity changes between these, or the appearance of new bands in a spectrum. The decision based on the polarised spectrum is independent of these factors. Another complementary technique used is dynamic light scattering (DLS), by which it is possible to obtain, for example, information regarding the macroscopic size (average diameter) of biological units. In DLS experiments, the intensity fluctuations of the elastically scattered light caused by the diffusion or Brownian motion of the scatterers are analysed. The intensity fluctuations can be converted into a size distribution by means of appropriate assumptions and mathematical tools as well as Mie theory.3 Aggregation of bio-molecules plays an important role in various bio-physical processes. The polarisation properties of RRS can reveal molecular changes such as aggregation and conformational changes; hence polarised Raman spectra can be used to study the degree of molecular aggregation in bio-molecules in their natural environment.

Revealing aggregated haemoglobin molecules in living erythrocytes

Haemoglobin (Hb) molecules with four oxygen binding sites assemble in erythrocytes in nearly all vertebrates. The aggregation of Hb molecules in living erythrocytes of human blood has been studied using polarised Raman scattering on spatially oriented erythrocytes combined with a principal component analysis (PCA).4 The conclusion reached was that a distinct ordering of haemoglobin took place within the erythrocytes. It is possible, however, to reach a similar conclusion regarding aggregation of intracellular Hb with a minimum of sample preparation and without orienting the erythrocytes. This can be achieved by taking advantage of the tensorial nature of the Raman process by measuring the polarised resonance Raman spectra of: (1) a dilute solution containing a number of randomly oriented erythrocytes (see Figure 1), and (2) a dilute reference solution containing a number of randomly oriented Hb molecules (not shown) and finally calculating the DPR values of the different modes (a2g modes) from these spectra. In RRS, there is no upper limit of the DPR value; it may be either a constant or depend on the excitation wavenumber (polarisation dispersion). Figure 2 shows a model calculation giving the DPR (a2g
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Hemoglobin(Hb)moleculeswith4oxygenbindingsitesassembleinerythrocytesinnearlyallvertebrates.TheaggregationofHb moleculesinlivingerythrocytesofhumanbloodhasbeenstudiedusingpolarizedRamanscatteringonspatiallyorientederythro 25 NO. 5 (2013) cytesVOL. combined with aprincipalcomponentanalysis(PCA)(4).Theconclusionreachedwasthatadistinctorderingofhemoglobin tookplacewithintheerythrocytes.Itis,however,possibletoreachasimilarconclusionregardingaggregationofintracellularHb withaminimumofsamplepreparationandwithoutorientingtheerythrocytes.Thiscanbeachievedbytakingadvantageofthe tensorialnatureoftheRamanprocessbymeasuringthepolarizedresonanceRamanspectraof:1)Adilutesolutioncontaininga dilutereferencesolutioncontaininganumberofrandomlyori numberofrandomlyorientederythrocytes(seefigure1)and2)A entedHbmolecules(notshown)andfinallycalculatingtheDPRvaluesofthedifferentmodes(a2gmodes)fromthesespectra.

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Excitationwave number

states,whichparticipateintheRRSprocess,willgiveriseton changesarecausedbyalterationsintheinterferencebetween theresonance Raman process. The aggregation of Hbwill ther Figure 1. Polarized Resonance Raman (perpendicularly polarized (black), parallel polarized (red), 532 nmexcitation) of a blood substitute due to, for example, the Figure 1. Polarised resonance Raman spectraspectra [perpendicularly polarmodes. the polarization dispersion ofthe dispersive a2gof ised (black), parallel polarised (red), 532nm excitation] of a solution solution containing randomly oriented erythrocytes inphosphate buffered saline (PBS). high resistance towards oxidation the
containing randomly oriented erythrocytes in phosphate buffered saline (PBS).

Figure2.TheDPR(a2gmode)ofanHbdimerasafunctionofthewavenumber.T Figure 2. The DPR (a2g mode) of an Hb dimer as a function of the Luckily,duetothenatureoftheRamanprocess,evensmallch wavenumber. The excitation wavenumber is 18.8kcm1.

mode) as a function of the wavenumber in the case of dimerisation of Hb. When an isolated haeme-group exhibits its ideal four-fold symmetry, the DPR of the a2g modes shows no dispersion and has the value infinity, but when encountering a symmetry lowering perturbation (for example, the incorporation into Hb), the DPR of these modes becomes dispersive and will decrease. In Reference 5, it is calculated that, for porcine Hb, the a2g mode n19 has the DPR value, DPRn19=71. From model calculations accounting for intermolecular interactions between Hb molecules (monomers), it follows that the excited states of these individual molecules, upon aggregation, will split into a number of states with slightly different energies, see Figure 3. Luckily, due to the nature of the Raman process, even small changes in the electronically excited states, which participate in the RRS process, will give rise to noticeable changes in the DPR. These changes are caused by alterations in the interference between the excited states participating in the resonance Raman process. The aggregation of Hb will, therefore, induce yet more changes in the polarisation dispersion of the dispersive a2g modes.
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haeme Fe ion. This is explained by the very complex, crowded and protected haeme environment. As opposed to the four oxygen-binding sites in vertebrate Hb, Hbl Hb has 144 haeme groups (or oxygen In practice, the DPR 1 value needs to binding sites) and is freely dissolved be even further decreased in order to within the vessels of the lugworm. The unequivocally conclude that the Hb giant molecule has a molecular mass molecules may be aggregated. In the in the mega Dalton regime, i.e. approxicase of aggregated Hb in the erythromately 4106Da.68 Biophysical cytes, the DPR value is DPRn19=31.5 and Hence, it is possible to Figure demonstrate an structural information aboutwill the giant 3.Ttheexcitedelectronicstates ofthemonomers upon aggregationo different energies. aggregation or perturbation by moni- protein can be disentangled by combining Inpractice, theDPR value needs tobe evenfurther decreased toring the polarisation properties in the the unique polarisation properties of RRS form of changes in the DPR values. with the size information obtained by DLS. thattheHbmoleculesmaybeaggregated.Inthecaseofaggre on dilute DPRvalueisDPR19=The 31DLS (5).measurements Hence,itispossible todemonst solutions of Hbl Hb, which provides an ofchang A study of extracellular bymonitoringthepolarizationpropertiesintheform average hydrodynamic diameter, d giant haemeoglobin in h, of A study of Extracellular Giant Hemoglobin in Areni Hbl Hb, were performed at several pH Arenicola marina based ized RRS and DLS. values.5 At pH=5 to 7, the Hbl Hb moleon polarised RRS and DLS cule exists in its native in which Extracellular haemoglobin (Hbl Hb) from Extracellular hemoglobin (Hbl Hb) fromform, Arenicola Marina(lugw case the average diameter, d Arenicola marina (lugworm) is considto resista ingcandidateasbloodsubstitutedueto,e.g., thehigh h, is equal ered to be a promising candidate as a 24.2nm. pH9, a dissociation process andpr Fe ion.This is explained byAt the very complex,crowded has taken place, resulting in dh=9nm, posedtothe4oxygen bindingsitesinvertebrateHb,HblHbh see Figure 4. At pH=10, however, only bindingsites)andis freely dissolvedwithinthevesselsofthel large aggregates, which consist of fragmolecularmassinthe mega Dalton regime, i.e.approximately mented Hbl Hb, with d h larger than andstructuralinformation the giantproteincanbedise 1000nm,about could be detected. polarizationproperties ofRRSwithof the size information obtain A comparison the DLS results for pH=10, with the behaviour of the TheDLSmeasurementsondilutesolutionsofHblHb,whichp RRS measurements (the DPR Hb,were performed atseveral pHvalues(5 diameterdhofHblpolarised values of three a2g modes), reveals that culeexistsinitsnativeform,forwhichcasetheaveragediame no further distortion of the haeme group dissociation process hastaken place resulting in dh= 9nm, see Figure3.Ttheexcitedelectronicstates ofthemonomers willuponaggregation ofthese splitinto anumberofstates with slightly took place upon aggregation. Hence, different3. energies. Figure The excited electronic states of the the coupling between the fragments or Inpractice,will, theupon DPRvalue needsto beevenfurther decreased inorder tounequivocally conclude monomers aggregation of these, that the Hb molecules may be aggregated. In the case of aggregated Hb in the erythrocytes, the aggregation did not involve any direct split into a number of states with slightly 2 DPRvalue isDPR19=31(5).Hence,itispossibleto demonstrate anaggregation orperturbation different energies. interaction between the haeme groups.
bymonitoringthepolarization propertiesintheformofchangesintheDPRvalues.

Extracellularhemoglobin(HblHb)fromArenicolaMarina(lugworm)isconsideredtobeapromis ingcandidateasbloodsubstitutedueto,e.g.,thehighresistancetowardsoxidationoftheheme Feion.Thisisexplainedbytheverycomplex,crowdedandprotectedhemeenvironment.Asop

A study of Extracellular Giant Hemoglobin in Arenicola Marina based on polar www.spectroscopyeurope.com ized RRS and DLS.

TheDLSmeasurementsondilutesolutionsofHblHb,whichprovidesanaveragehydrodynamic VOL. 25 NO. 5 (2013) Hbl Hb mole diameterdhofHblHb,wereperformedatseveralpHvalues(5).AtpH=5to7the culeexistsinitsnativeform,forwhichcasetheaveragediameterdhisequalto24.2nm.AtpH9a dissociationprocesshastakenplaceresultingindh=9nm,seefigure4.AtpH=10,however,only largeaggregates,whichconsistoffragmentedHblHb,withdhlargerthan1000nmcouldbede tected.

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sensitised solar cells is reported. One of the key parameters governing the longterm thermal stability of a dye-sensitised solar cell is, of course, the dye stability, i.e. whether or not this is degraded by thermal stress.

Acknowledgement
K.D. Jernshj greatly acknowledges support from Energi Fyn. sites)andisfreelydissolvedwithinthevesselsofthelugworm.ThegiantmoleculehasamolecularmassinthemegaDaltonre 6 thegiantproteincanbedisentangledby gime,i.e.approximately4x10 Da(6)(7)(8).Biophysicalandstructuralinformationabout References combiningtheuniquepolarizationpropertiesofRRSwiththesizeinformationobtained DLS. The Raman Effect: A Unified 1. by D.A. Long, Treatment of the of Raman ofHblHb, wereper TheDLSmeasurementsondilutesolutionsofHblHb,whichprovidesanaveragehydrodynamic diameter dhTheory Scattering by Molecules. John Wiley & formedatseveralpHvalues(5).AtpH=5to7theHblHbmoleculeexists initsnativeform, forwhichcasetheaveragediameterdh Sons Ltd, Chichester, UK (2002). equal to24.2 pH9adissociation process has taken place resulting indh=9nm,seefigure4.AtpH=10,however,only Figure 4. is DLS particle (Hblnm. Hb)At distributions, i.e. the relative number of particles versus particle than 1000 nmcould S. beHassing detected. =O. large aggregates, which consist offragmented Hbl Hb, to with d diameter pH 5 (native Hbl Hb) and 9 (fragments of Hbl Hb due dissociation). Each 2. and Sonnich Mortensen, Figurefor 4.DLS = particle (Hbl Hb) distributions, i.e.the relative number of particles versus particle diameter forpH 5(native Hbl Hb)and9 hlarger Advances in Infrared Raman distribution contains three measurements coloured blue, green Reproduced with Acomparison the DLS results for pH = 10 withand the purple. behavior ofthe polarized RRS measurements (the DPR values ofthree a2g (fragments ofHbl Hbof due to dissociation). Each distribution contains three measurements colored blue,green and purple. and Reproduced Spectroscopy, Vol. 6: Polarization and permission from J. Chem. Phys . 139, 065104 (2013). Copyright Institute modes) reveals that nofurther distortion of theheme American group took placeof upon aggregation. Hence, betweenthe frag withpermission fromJ. Chem. Phys. 139, 065104 (2013). 2013 Copyright 2013 American Institute ofPhysics. thecoupling Interference Phenomena in Resonance Physics. mentsoraggregationdidnotinvolveanydirectinteractionbetweenthehemegroups. Raman Scattering . Heyden and Sons, AcomparisonoftheDLSresultsforpH=10withthebehaviorofthepolarized RRSmeasurements London, UK (1980).

(theDPRvaluesofthreea2gmodes)revealsthatnofurtherdistortionofthehemegrouptook 3. R. Pecora (Ed), Dynamic Light Scattering, placeuponaggregation.Hence,thecouplingbetweenthefragmentsorApplications aggregation notCorrelation in of did Photon Spectroscopy. Plenum Press, New York, volveanydirectinteractionbetweenthehemegroups. USA (1985).
4. B . R . W o o d , L . H a m m e r a n d D . McNaughton, Resonance Raman spectroscopy provides evidence of heme ordering within the functional erythrocyte, Vib. Spectrosc. 38(12), 71 (2005). doi: 10.1016/j.vibspec.2005.02.016

5. K.D. Jernshj, S. Hassing and L.F. Olsen, A combination of dynamic light scattering and polarized resonance Raman scattering applied in the study of Arenicola marina extracellular hemoglobin, J. Chem. Phys. 139(6), 065104 (2013). doi: Figure 5. In Polarised spectra [parallel polarised (red) and perpendicularly (blue)] of thehemein of nativeHblHbandporcineHb addition, an analysis of the DPR ofthreepolarised a2gmodes 10.1063/1.4813920 native Hbl Hb from Arenicola marina (pH=5), obtained with 532nm excitation. Reproduced Figure 5polarized spectra (parallel polarized (red) and perpendicularly polarized(blue))ofnativeHblHbfromArenicolaMarina 6. L.M.139, Moreira, A.L. Poli, J.P. Lyon, F.V. with permission from J. Chem. Phys . 139, 065104 (2013). Copyright 2013 American Institute of J.Chem. (pH=5), obtained with 532 nm excitation. Reproduced with permission from Phys. 065104 (2013). Copyright 2013 Santos, P.C.G. Moraes, J.P.F. Mendona, Physics. AmericanInstituteofPhysics. V.C. Barbosa and H. Imasato, Giant extracellular hemoglobin of Glossoscolex showsthatthedistortionofthehemegroupawayfromitsidealfourfold symmetryissmallerfor paulistus: excellent prototype of biosenabsorption polarized spectra (blue)) of In addition, an analysis of the DPR of Figure5polarized spectra (parallel polarizedThe (red)visible andperpendicularly ofnativeHblHbfromArenicolaMarina sor and blood substitute, Chapter 18, in heme groups bound in the lugworm Hbl Hb (i.e. DPR = 15 2) than for heme groups bound in 19 Hb, erythrocytes Hbl Hb Phys.139, three a of the haeme native (pH=5), obtained with 532in nm excitation.porcine Reproduced with permissionand from J.Chem. 065104 (2013). Copyright 2013and 2g modes Biosensors for Health, Environment porcine Hb (DPR = 1).To summarize: American Institute of Physics. 7 (pH=5 and 10) are Hbl Hb and porcine Hb shows that the Biosecurity. InTech Open Access Publisher 19 4alike, except for (2011). minor differences not relevant here, distortion of the haeme group away from Molecule DPR19 which indicates that the molecuits ideal four-fold symmetry is smaller for 7. M. Tabak, F.A.O. Carvalho, J.W.P. Carvalho, hemegroup lar configuration of the haeme group haeme groups bound in the lugworm J.F.R. Bachega and P.S. Santiago, Recent Hb 71 characterizations on the giant extracorresponding to the excited state is Hbl Hb (i.e. DPRn19=152) than for porcine cellular hemoglobin of Glossoscolex not strongly perturbed. By analysing the haeme groups bound in porcine Hb porcine Hbaggregatedinerythrocytes paulistus 31 and some other giant hemoproperties, it is possible to (DPRn19=71). To summarise: globins Hblpolarisation Hb 15 2from different worms, Chapter 15, in Stoichiometry and ResearchThe gain additional molecular information Importance of Quantity in Biomedicine, DPRn19 Molecule sometimes barely accesible using the InTech Open Access Publisher (2012).

Thevisibleabsorption spectra ofporcine Hb,erythrocytesandHblHb(pH=5and10)arealike unpolarised spectra. 8. J. the Elmer and A.F. Palmer, Biophysical of forminor differencesnotrelevanthere,whichindicatesthat molecular configuration Porcineexcept Hb 71 properties of Lumbricus terrestris erythOutlook rocruorin and Its potential use asthe a red group corresponding totheexcitedstateisnotstronglyperturbed. By analyzing Porcinethe Hbheme aggregated 31 In a forthcoming publication, a resoblood cell substitute, Open Access J. in erythrocytes polarizationpropertiesitispossible togainadditionalmolecularinformation sometimes barely Funct. Biomater . 3(1), 49 (2012). doi: nance Raman-based polarisation study 10.3390/jfb3010049 Hbl Hb accesibleusingthe 152 unpolarized spectra. of the thermal stability of a dye from dyeOutlook SPECTROSCOPYEUROPE 15 www.spectroscopyeurope.com InaforthcomingpublicationaresonanceRamanbasedpolarizationstudy ofthethermalstability ofadyefromdyesensitizedsolarcellsisreported.Oneofthekeyparametersgoverningthelong termthermalstabilityofadyesensitizedsolarcellisofcoursethedyestability,i.e.whetherornot
Haeme group