Novel Chiroptical Analysis of Hemoglobin by Surface Enhanced Resonance Raman Optical Activity Spectroscopy NADEZDA A. BRAZHE, 1 ALEXEY R. BRAZHE, 1 OLGA V. SOSNOVTSEVA, 2,3 AND SALIM ABDALI 4 * 1 Department of Biophysics, Biological Faculty, Moscow State University, Moscow, Russia 2 Department of Biomedical Sciences, Copenhagen University, Copenhagen N, Denmark 3 Department of Physics, Technical University of Denmark, Lyngby, Denmark 4 Molecular cancer biology, Danish Cancer Society, Copenhagen Ø, Denmark Contribution to the Special Thematic Project ‘‘Advances in Chiroptical Methods’’ ABSTRACT The metalloprotein hemoglobin (Hb) was studied using surface enhanced resonance Raman spectroscopy (SERRS) and surface enhanced resonance Raman optical activity (SERROA). The SERROA results are analyzed and compared with the SERRS, and the later to the resonance Raman (RRS) performed on Hb. The SERRS measurements careful optimization, with respect to the concentration and vol- ume ratio of the analyte to colloids, enables for the first time SERROA of this molecule. We observed that the most intense SERROA signals were attributed the m 4 , m 20 , and m 21 vibrations, which are sensitive to the redox state of the heme’s iron ion, and to the pres- ence of its sixth site, bound to exogenous ligand; O 2 , NO or CO. However, in this study, the SERROA signals corresponding to these vibrations appear more sensitive to the Hb oxygen-binding properties than they appear in the SERRS or RRS. Moreover, the SER- ROA signal of Hb has successfully been monitored as a function of time, and was observed to be stable for 4–5 min. To our knowledge, the SERROA results of Hb, and its comparison to SERRS and RRS, are here reported for the first time. Chirality 21:S307–S312, 2009. V V C 2010 Wiley-Liss, Inc. KEY WORDS: SEROA/SERROA; chirality; hemoglobin; resonance raman spectroscopy; SERS/SERRS INTRODUCTION Raman optical activity (ROA) is a vibrational spectro- scopical tool, which mainly determines the chirality of mol- ecules by measuring the tiny difference in the Raman scat- tered signal by right-circular and left-circular polarized light. This tool has shown to be more sensitive to the backbone of a molecule than the side chains, whereas con- ventional Raman on the other hand is very sensitive for. Therefore, ROA and Raman scattering are considered complementary in full studies of molecules, especially in biology. 1,2 However, in spite of its wealth of information, provided for the folding, secondary and tertialy structures, ROA remains less widely spread than Raman, because the ROA signal is three to four orders of magnitude weaker than the Raman signal. This is because of the measured in- tensity difference, which is called the circular intensity dif- ference, CID, usually referred to as the dimensionless D. The surface enhanced Raman scattering (SERS) on the other hand provides much stronger signal, when a mole- cule is found in the vicinity of a nanoparticle. SERS was observed by Fleischmann and co-workers. 3 on pyridine adsorbed on a roughened silver surface, and it was first identified independently by Jeanmaire and Van Duyne, 4 who attributed it the charge transfer taking place between the molecule and the nanoparticle, and Albrecht and Creighton 5 , who described it because of the electromag- netic effect. SERS has during the last couple of decades been applied for all kinds of samples, making use of its capability of measuring and detecting structural changes in concentrations below the micromolar level. 6–10 In the following study, we succeeded to combine the SERS method, using colloid solution of silver nanoparticles (Ag NPs), with ROA. The combination of these two meth- ods, into surface enhance Raman optical activity (SEROA), was first treated theoretically by Efrima 11,12 who has shown that such combination could be applicable, and enhancement could be achieved in the chiral signal. In spite of these studies and the following theoretical devel- opments of the method, 13–16 no reliable experimental study has verified the method, although two reports 17,18 have addressed SEROA, but the results were not convinc- Contract grant sponsor: Russian Foundation for Basic Research; Contract grant numbers: 070400621, 080400531, 090400646, 090312221. Contract grant sponsor: Russian Federation for 2008–2010; Contract grant number: 5, 2008-3-3.1-049. Contract grant sponsor: Danish National Research Foundation. *Correspondence to: Salim Abdali, Danish Cancer Society, Strandboule- varden 49, Copenhagen Ø, Denmark. E-mail: abdali@cancer.dk Received for publication 12 May 2009; Accepted 5 November 2009 DOI: 10.1002/chir.20820 Published online 4 January 2010 in Wiley InterScience (www.interscience.wiley.com). CHIRALITY 21:S307–S312 (2009) V V C 2010 Wiley-Liss, Inc.