Application Of Resonance Raman Spectroscopy Research Articles

Probing the structure-function relationship of hemoglobin in living human red blood cells.

Hemoglobin (Hb) is a key component of respiratory system and as such plays important role in human physiology. The studies of Hb's structure and functions are usually performed on cell-free protein; however, it has been shown that there are functionally relevant differences between isolated Hb and Hb present inside red blood cells (RBCs). It is clear that new experimental approaches are needed to understand the origin of these differences and to gain insight into the structure-function relationship of Hb within intact living cells. In this work we present a novel application of Resonance Raman spectroscopy to study heme active site of different forms of human Hb within living RBCs using laser excitation lines in resonance with their Soret absorption bands. These studies revealed that there are no significant changes in the disposition of the Fe-O-O fragment or the Fe-NHis linkage for Hb molecules enclosed in RBCs and these in free isolated states. However, some changes in the orientation of the heme vinyl groups were observed which might account for the differences in the protein activity and ligand affinity. This work highlights importance of protein-based studies and presents a new opportunity to translate these results to physiological cell systems.

Raman spectroscopy analysis of molecular configuration forms of the macular xanthophylls

AbstractMacula lutea, the yellow spot of the retina of the human eye, comprises three xanthophyll pigments: lutein, zeaxanthin, and meso‐zeaxanthin, playing numerous important biological functions. Macular xanthophylls are exposed to relatively strong illumination in the eye, owing to the fact that the yellow spot is localized on the optical axis, in the frontal layer with respect to photoreceptors. In the present work, we address the problem of photostability and possible photoisomerization of macular xanthophylls with the application of resonance Raman spectroscopy. The results show photostability of the two major isomers of the macular xanthophylls, all‐trans and 9‐cis, and efficient photoconversion of the 13‐cis isomer to the 9‐cis and mostly to the all‐trans form. We report the Raman spectra of the main molecular configuration forms of the macular xanthophylls, opening an avenue for the examination of their possible presence and photoconversion in natural systems.

111 - Application of resonance raman spectroscopy on manganese porphyrin detection and redox state in endothelial cells: implications on cytoprotection towards peroxynitrite

Manganese(III) ortho N-substituted pyridylporphyrins (MnP) act as efficient antioxidants catalyzing superoxide dismutation and accelerating peroxynitrite reduction. Importantly, MnP can reach mitochondria offering protection against reactive species in different animal models of disease. In this scenario, we applied Resonance Raman (RR) spectroscopy as a direct method to detect both the uptake and the redox state of MnP in living cells. Using RR we were able to analyze the intracellular redox state changes of two potent MnP (MnTnBuOE-2-PyP5+ and MnTnHex-2-PyP5+) within endothelial cells exposed to SIN-1. RR experiments with cells and isolated mitochondria revealed that the reduction of Mn(III)P was affected by inhibitors of the electron transport chain, supporting the action of MnP as efficient redox active compounds in mitochondria. Indeed, RR spectra confirmed that MnP added in the Mn(III) state can be incorporated into the cells, readily reduced by intracellular components to the Mn(II) state and oxidized by peroxynitrite. We also evaluated the cytoprotective capacity of MnP by exposing the endothelial cells to nitro-oxidative stress induced by peroxynitrite. We observed a preservation of normal mitochondrial function, attenuation of cell damage and prevention of apoptotic cell death. These data introduce a novel application of RR spectroscopy for the direct detection of MnP and their redox states inside living cells, and helps to rationalize their antioxidant capacity in biological systems.

P 095 - Application of Resonance Raman spectroscopy for the direct detection of manganese porphyrins and their redox state in endothelial cells

Manganese porphyrins (MnPs) can act as efficient catalytic antioxidants. MnPs can reach mitochondria and confer pharmacological protection in different models of disease where oxidants such as peroxynitrite participate. MnPs quantitation and subcellular distribution by LC-MS/MS has been reported; however, a direct method to evaluate their uptake and redox state in living cells has not been developed. Here, we applied Resonance Raman (RR) confocal microscopy and confirmed that the MnPs-based lipophilic SOD mimics, MnTnBuOE-2-PyP5+ and MnTnHex-2-PyP5+ were incorporated into endothelial cells, and that Mn(III)P can be reduced by intracellular components such as the mitochondrial electron transport chain. Experiments with isolated mitochondria revealed the reduction to Mn(II)P, which was in part affected by the inhibitors of electron transport chain, supporting the action of MnPs as efficient redox active compounds in mitochondria. We have also characterized the reaction kinetics of MnTnBuOE-2-PyP5+ with peroxynitrite and evaluated the cytoprotective capacity of MnPs by exposing the endothelial cells to nitro-oxidative stress induced by peroxynitrite. This data introduce a novel application of the RR spectroscopy for the direct detection of MnPs and their redox state in living cells, and helps to rationalize its antioxidant capacity in biological systems.

Application of resonance Raman spectroscopy for interrogation of cryoradiolytically reduced oxygenated heme proteins

Resonance Raman (rR) spectroscopy is generally useful to interrogate unstable intermediates that arise in the enzymatic cycles of heme enzymes, such as peroxo-, hydroperoxo- and ferryl species. While it is typically difficult to trap these elusive intermediates in solution, the cryoradiolysis approach offers a way to successfully overcome these obstacles. The present work employs modified derivatives of horse heart myoglobin to demonstrate the methodology and utility of a combination of rR and cryoradiolysis for detection and structural characterization of Fe―O―O, Fe―O―O―H and FeO fragments of such reactive species. First, the effect of active site mutations on the Fe―O―O fragments was investigated; specifically, the His64Leu, Val68Ser and Ile107Ala in the distal pocket and Ser92Ala, Ser92Leu and His97Phe on the proximal side. The current work has permitted documentation of the wavenumbers of the ν(Fe―O) stretching modes of the relatively unstable dioxygen adducts of these mutants, some of them previously unreported. Significantly, applying the cryoradiolysis approach has yielded previously unavailable vibrational spectroscopic data for the peroxo-, hydroperoxo and ferryl derivatives of these mutants. Of the six mutations investigated, only two, the H64L and V68S, replacements had a significant effect on the structure and stability of these unstable intermediates. In addition to these manipulations of the heme active site environment, the study of myoglobins bearing modified hemes was also undertaken; namely, myoglobins reconstituted with mesoheme and diformyl deuteroheme. The results show that the heme equatorial electronic effects significantly influence the stability and internal modes of the Fe―O―O and FeO fragments of these unstable oxy intermediates. Copyright © 2016 John Wiley & Sons, Ltd.

Application of Resonance Raman Spectroscopy in the Characterization of Single-Walled Carbon Nanotubes

Application of Resonance Raman Spectroscopy in the Characterization of Single-Walled Carbon Nanotubes

Elucidating photoinduced structural changes in phytochromes by the combined application of resonance Raman spectroscopy and theoretical methods

Phytochromes constitute a family of red-light sensing photoreceptors in plants and microorganisms. The photoactive cofactor is an open-chain methine-bridged tetrapyrrole that, upon light absorption, undergoes a double bond isomerisation followed by series thermal relaxation processes which eventually lead to the functional structural change of the protein. Resonance Raman spectroscopy has contributed significantly to the understanding of the molecular functioning of these proteins although both the experiments and the interpretation of the spectra represent a considerable challenge. This account is dedicated to describe achievements, potential and limitations of combined resonance Raman spectroscopic and theoretical approaches for elucidating cofactor structures in phytochromes. Experimental approaches are discussed paying specific attention on strategies to overcome unwanted photochemical and photophysical processes when probing the various states of the photoinduced reaction cycle of phytochromes. The most comprehensive set of experimental data on phytochromes, including engineered protein variants and adducts formed with isotopically labelled tetrapyrroles, has been obtained by resonance Raman spectroscopy with near-infrared excitation that also allows probing phytochrome crystals without photo-induced destruction. Quantum mechanical calculations of Raman spectra of model compounds represent a first approximation for determining the methine bridge geometry of the protein-bound tetrapyrroles and constitute the basis for the identification of marker bands for specific structural properties such as the protonation state of the cofactor. Drawbacks of this theoretical method that inevitably neglects the protein environment have become evident with the first determinations of three-dimensional structures of phytochromes. These structural models can now be used for employing hybrid methods that combine quantum mechanical and molecular mechanics calculations of the cofactor and the protein matrix, respectively. Although further developments are required for a more accurate description of the protein force field, this methodology promises to become a powerful tool for a comprehensive extraction of structural information from the Raman spectra such it may allow refining structural models of the cofactor site derived by protein crystallography.

Defining the Structural Consequences of Mechanism-Based Inactivation of Mammalian Cytochrome P450 2B4 Using Resonance Raman Spectroscopy

In view of the potent oxidizing strength of cytochrome P450 intermediates, it is not surprising that certain substrates can give rise to reactive species capable of attacking the heme or critical distal-pocket protein residues to irreversibly modify the enzyme in a process known as mechanism-based (MB) inactivation, a result that can have serious physiological consequences leading to adverse drug-drug interactions and toxicity. While methods exist to document the attachment of these substrate fragments, it is more difficult to gain insight into the structural basis for the altered functional properties of these modified enzymes. In response to this pressing need to better understand MB inhibition, we here report the first application of resonance Raman spectroscopy to study the inactivation of a truncated form of mammalian CYP2B4 by the acetylenic inhibitor 4-(tert-butyl)phenylacetylene, whose activated form is known to attach to the distal-pocket T302 residue of CYP2B4.

Application of Resonance Raman Spectroscopy to Electronic Structure in Metal Complex Excited States. Excited-State Ordering and Electron Delocalization in Dipyrido[3,2-a:2',3'-c]phenazine (dppz): Complexes of Re(I) and Ru(II)

Excited-state ordering and electron delocalization in Dipyrido[3,2-a:2`,3`-c]phenazine (dppz): Complexes of Re(I) and Ru(II). From excitation-dependent, resonance Raman measurements on [Ru(dmb){sub 2}(dppz)]{sup 2+} (dmp is ,4`-dimethyl-2,2`-bipyridine; dppz is dipyrido [3,2-a:2`,3`-c]phenazine) in CH{sub 3}CN a metal-to-metal ligand charge transfer (MLCT) band at 450 nm is Ru{sup II} (d{pi}){r_arrow} dmb ({pi}){sup *} in character with Ru{sup II}(d{pi}){r_arrow}dppz ({pi}){sup *} appearing as a shoulder at {approx} 490 ligand and point to delocalization of the excited electron over the entire ligand framework. From transient resonance Raman measurements on fac-[Re(dppz)(CO){sub 3-}(PPh{sub 3})] + (PPh{sub 3} is triphenylphosphine) and fac-[Re(dppz)(CO){sub 3-}Cl], the lowest excited state in both is dppz-localized {sup 3}{pi}{pi}{sup *} in character even though the chloro complex is a MLCT emitter.

A Simple Mixer/Jet Cell for Raman Spectroscopic Studies

In biological applications of resonance Raman spectroscopy it is frequently desirable to reduce the instantaneous and long-term power density of the focused laser beam in order to preserve the sample. Both flowing sample methods and laser beam defocusing techniques have been used successfully by various groups to minimize damage to photolabile samples. The use of flowing sample cells under conditions in which sample recycling is practical has the additional advantage that long acquisition times can be achieved with fairly minimal sample consumption. Nonetheless, the flowing cells described to date are most useful when both fairly large volumes of sample are available and recirculation is feasible. Recirculation is often not possible if the sample of interest is an unstable reaction intermediate, and such an application requires rapid mixing of the precursor compounds. A further consideration with Raman flow cells involves the sample containment technique in the scattering volume. Quartz capillaries are often used, but with these, quartz scattering is severe in the low-frequency region, where it overlaps vibrational modes of the sample and makes their detection difficult. Several groups have avoided this problem by arranging their flowing cells so that the sample forms a free jet in air in the scattering volume. A notable example of this is the microdroplet mixing technique developed by Kincaid and co-workers that uses continuous wave excitation and both rapid mixing and Raman scattering in air.

Resonance Raman spectroscopy of single crystals of bacterial cytochrome c4

The application of resonance Raman spectroscopy to single crystals of cytochrome c 4 from Pseudomonas aeruginosa is described. The technique is shown to be a suitable method of distinguishing between the oxidised and reduced forms of the protein. This is particularly useful when the crystal is to be used for X-ray diffraction data collection. Cytochrome c 4 Oxidation state Resonance Raman spectroscopy Single crystal spectroscopy

Resonance raman spectra and photochemical reactivity of transition metal α-diimine complexes

In this article the application of resonance Raman spectroscopy to the study of metal to ligand charge transfer (MLCT) transitions of α-diimine complexes is described. From these spectra information is obtained about the character of the MLCT transitions and about the properties of the excited states. It is shown how these resonance Raman spectra can be used to interpret and predict the MLCT photo-chemistry of α-diimine and imine complexes.

The application of resonance raman spectroscopy to the study of the vibrational and electronic properties of inorganic species

The application of resonance Raman spectroscopy to the study of the vibrational and electronic properties of molecular inorganic species, metal-metal bonded complexes, oxygen- nitrogen- and dinitrogen bridged complexes, and to linearchain halogen-bridged complexes is described, and shown to yield valuable and, in many cases, unique molecular information.

Biological applications of resonance Raman spectroscopy: haem proteins

Resonance Raman spectroscopy involves laser excitation within an absorption band of the sample. Certain vibrational modes, those which couple to the electronic transition, exhibit greatly increased Raman scattering in the resulting spectrum. Sensitivity approaches that of absorption spectrophotometry, while the high resolution characteristic of vibrational spectroscopy is preserved, even in solution at room temperature. If the resonant electronic transition is associated with a site of biological activity, then the technique offers a sensitive probe for structural features of the site. Haem proteins afford particularly informative resonance Raman spectra, with a rich assortment of porphyrin ring vibrations, which can be classified and analysed via their symmetry properties. Certain of these frequencies are sensitive to the structural concomitants of spin- and oxidation-state changes of the haem group. These can be used to monitor the structural consequences of ligation or electron transfer in haem proteins.

Biochemical Applications of Infrared and Raman Spectroscopy

Uses to which infrared and Raman spectroscopy have been put in biochemical work are reviewed. In the infrared field some of the applications discussed are hydrogen bonding, carbohydrates, lipids, proteins and polypeptides, hydrogen-deuterium exchange, nucleic acids, steroids, porphyrins, enzymology, and medical uses, among others. In the Raman field some of the applications discussed are polypeptides, proteins and enzymes, nucleic acids, carbohydrates, steroids, membranes, lipids, and calcified tissues. Some applications of resonance-Raman spectroscopy are discussed. They include such substances as carotenoids and plant pigments, enzymes, hemoglobin, cytochrome c, hemocyanin, visual pigments, and protein-ligand and antibody-hapten interactions. The kinds of information obtainable with each of the methods and the various classes of biochemical substances are given in some detail, and pertinent references are supplied.