ABSTRACT
The article presents a noninvasive approach to the study of erythrocyte properties by means of a comparative analysis of signals obtained by surface-enhanced Raman spectroscopy (SERS) and resonance Raman spectroscopy (RS). We report step-by-step the procedure for preparing experimental samples containing erythrocytes in their normal physiological environment in a mixture of colloid solution with silver nanoparticles and the procedure for the optimization of SERS conditions to achieve high signal enhancement without affecting the properties of living erythrocytes. By means of three independent techniques, we demonstrate that under the proposed conditions a colloid solution of silver nanoparticles does not affect the properties of erythrocytes. For the first time to our knowledge, we describe how to use the SERS-RS approach to study two populations of hemoglobin molecules inside an intact living erythrocyte: submembrane and cytosolic hemoglobin (Hb(sm) and Hb(c)). We show that the conformation of Hb(sm) differs from the conformation of Hb(c). This finding has an important application, as the comparative study of Hb(sm) and Hb(c) could be successfully used in biomedical research and diagnostic tests.
Subject(s)
Erythrocytes/chemistry , Spectrum Analysis, Raman , Animals , Cell Survival , Cytosol/metabolism , Erythrocytes/cytology , Erythrocytes/drug effects , Erythrocytes/metabolism , Gold/chemistry , Gold/pharmacology , Hemoglobins/chemistry , Hemoglobins/metabolism , Male , Metal Nanoparticles , Rats , Rats, Wistar , Signal Processing, Computer-Assisted , Surface PropertiesABSTRACT
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 volume 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 nu(4), nu(20), and nu(21) vibrations, which are sensitive to the redox state of the heme's iron ion, and to the presence 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 SERROA 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.
Subject(s)
Hemoglobins/analysis , Research Design , Spectrum Analysis, Raman/methods , Animals , Colloids , Heme/chemistry , Hemoglobins/chemistry , Male , Optical Rotation , Oxidation-Reduction , Rats , Rats, Wistar , VibrationABSTRACT
Raman optical activity (ROA) directly monitors the stereochemistry of chiral molecules and is now an incisive probe of biomolecular structure. ROA spectra contain a wealth of information on tertiary folding, secondary structure and even the orientation of individual residues in proteins and nucleic acids. Extension of ROA to an even wider range of samples could be facilitated by coupling its structural sensitivity to the low-concentration sensitivity provided by plasmon resonance enhancement. This leads to the new technique of surface enhanced ROA, or SEROA, which is complementary to both SERS and ROA. In this tutorial review, we present a survey of theoretical and experimental work undertaken to develop SEROA and discuss these efforts in the context of the ROA technique, and, based on the authors' work, outline possible future directions of research for this novel chiroptical spectroscopy.
Subject(s)
Molecular Probes/chemistry , Spectrum Analysis, Raman , Molecular Conformation , Optical Rotation , Spectrum Analysis, Raman/methods , Surface PropertiesABSTRACT
High-resolution resonance Raman (RR) and resonance Raman optical activity (ROA) spectra of cytochrome c were obtained in order to perform full assignment of spectral features of the resonance ROA spectrum. The resonance ROA spectrum of cytochrome c revealed a distinct spectral signature pattern due to resonance enhanced skeletal porphyrin vibrations, more pronounced than any contribution from the protein backbone. Combining the intrinsic resonance properties of cytochrome c with the surface plasmon enhancement achieved with colloidal silver particles, the surface enhanced resonance Raman scattering (SERRS) and surface enhanced resonance ROA (SERROA) spectra of the protein were successfully obtained at concentrations as low as 1 microM. The assignments of spectral features were based on the information obtained from the RR and resonance ROA spectra. Excellent agreement between RR and SERRS spectra is reported, while some disparities were observed between the resonance ROA and SERROA spectra. These differences can be ascribed to perturbations of the physical properties of the protein upon adhesion to the surface of the silver colloids.
