Raman spectroscopy in biomedicine – non-invasive in vitro analysis of cells and extracellular matrix components in tissues

Raman spectroscopy is an established laser-based technology for the quality assurance of pharmaceutical products. Over the past few years, Raman spectroscopy has become a powerful diagnostic tool in the life sciences. Raman spectra allow assessment of the overall molecular constitution of biological samples, based on specific signals from proteins, nucleic acids, lipids, carbohydrates, and inorganic crystals. Measurements are non-invasive and do not require sample processing, making Raman spectroscopy a reliable and robust method with numerous applications in biomedicine. Moreover, Raman spectroscopy allows the highly sensitive discrimination of bacteria. Rama spectra retain information on continuous metabolic processes and kinetics such as lipid storage and recombinant protein production. Raman spectra are specific for each cell type and provide additional information on cell viability, differentiation status, and tumorigenicity. In tissues, Raman spectroscopy can detect major extracellular matrix components and their secondary structures. Furthermore, the non-invasive characterization of healthy and pathological tissues as well as quality control and process monitoring of in vitro-engineered matrix is possible. This review provides comprehensive insight to the current progress in expanding the applicability of Raman spectroscopy for the characterization of living cells and tissues, and serves as a good reference point for those starting in the field.     

Identification of enzyme coupling sites with aromatic diazonium salts-a resonance raman study.

The coupling reaction of diazonium salts of aromatic compounds with the aromatic residues of proteins results in chromophoric covalent derivatives which yield strong resonance enhanced Raman spectra. The protein residues modified by these coupling reactions have been identified using the ν(NN) and ν(N-φ) vibrational bands in the resonance Raman spectra. Previous studies have established that diazoarsanilic acid couples with carboxypeptidase at tyrosine 248. The resonance Raman spectrum of arsanilazocarboxypeptidase was compared with spectra of arsanilazotyrosine and arsanilazohistidine model compounds; the results are consistent only with coupling at a tyrosine residue. This confirmation of the previously established site of modification establishes the utility of resonance Raman spectroscopy as a tool for identification of the site of covalent modification. To further investigate this approach, the diazonium salt of sulfanilamide (a site-specific reagent) was used to prepare a covalent coupling derivative of bovine carbonic anhydrase. The coupling reaction appears to have a stoichiometry of 1:1 and results in nearly complete loss of sulfanilamide binding capability and esterase activity. Comparison of the pH dependence of the resonance Raman spectra of sulfanilazocarbonic anhydrase with the spectra of sulfanilazotyrosine, sulfanilazohistidine, and sulfanilazotryptophan suggests that histidine is the site of modification of this new carbonic anhydrase derivative.     

Dynamics and reactivity of HbXL99 alpha. A cross-linked hemoglobin derivative

Resonance Raman spectroscopy, transient absorption, and fluroescence techniques have been employed to investigate the structure and dynamics of the alpha-cross-linked hemoglobin derivative, HbXL99 alpha. The resonance Raman spectra of the deoxy form of HbXL99 alpha are identical to those of native NbA (VFe-His approximately 222 cm-1), which exhibit a T-state (low affinity) structure regardless of solvent conditions. The resonance Raman spectra of the transient heme photoproduct resulting from CO photolysis from HbXL99 alpha appear to have structures intermediate between deoxy-T and ligand-bound R structures (VFe-His approximately 222 cm-1). Time-resolved resonance Raman data of HbXL99 alpha-CO show that complete CO recombination occurs after approximately 5 ms, with only a small amount of the CO-bound species reforming within approximately 200 ns (geminate recombination). Transient absorption spectra of HbXL99 alpha-O2 indicate that the extent of sub-nanosecond geminate recombination of O2 is also reduced in the cross-linked derivative relative to native HbA. The decrease in tryptophan fluorescence of HbXL99 alpha upon oxygenation further indicates that tertiary structural changes at the alpha 1-beta 2 interface upon ligation are apparently reduced, but not eliminated in the cross-linked derivative relative to HbA.     

Resonance Raman spectroscopy

Resonance Raman spectroscopy may yield precise information on the conformation of, and on the interactions assumed by, the chromophores involved in the first steps of the photosynthetic process, whether isolated in solvents, embedded in soluble or membrane proteins, or, as shown recently, in vivo. By making use of this technique, it is possible, for instance, to relate the electronic properties of these molecules to their structure and/or the physical properties of their environment, or to determine subtle changes of their conformation associated with regulatory processes. After a short introduction to the physical principles that govern resonance Raman spectroscopy, the information content of resonance Raman spectra of chlorophyll and carotenoid molecules is described in this review, together with the experiments which helped in determining which structural parameter each Raman band is sensitive to. A selection of applications of this technique is then presented, in order to give a fair and precise idea of which type of information can be obtained from its use in the field of photosynthesis.     

Sulfmyoglobin. Resonance Raman spectroscopic evidence for an iron-chlorin prosthetic group

The green heme protein sulfmyoglobin (SMb) has been suggested to contain a sulfur-modified iron chlorin prosthetic group. To evaluate this hypothesis, we have obtained high-frequency (greater than 1000 cm-1) resonance Raman spectra of both oxidized and reduced SMb with 457.9-, 488.0-, 514.5-, 568.2-, and 647.1-nm excitation. The SMb spectra are compared to those of native met- and deoxymyoglobin (Mb). Vibrational frequencies for SMb are generally similar to those of Mb, suggesting a high-spin state for both the Fe(III) and Fe(II) SMb species, as is typical of native Mb. However, major differences between SMb and Mb occur both for patterns of relative spectral intensities and for depolarization ratios. In particular, all B1g-depolarized porphyrin modes in the Mb spectra have become polarized, totally symmetric vibrational modes in the SMb spectra. These contrasts reflect a dramatic lowering of the effective symmetry for the SMb prosthetic group. Several new bands are observed in SMb spectra that are not present in spectra of either native Mb or iron protoporphyrin IX complexes. The observation of additional polarized bands flanking the oxidation state marker, V4, is of particular interest. In a parallel study, we compared the resonance Raman spectral properties of iron protoporphyrin IX-derived chlorins and metallo-octaethylchlorins with those of the analogous porphyrins: the chlorin spectra exhibited altered intensity patterns, an increased number of totally symmetric (polarized) vibrational bands, and several new vibrational bands, including one or two in the region of the oxidation state marker, V4. Thus, the resonance Raman spectral characteristics of SMb and metallo-chlorins are complementary and strongly support a chlorin prosthetic group for SMb. Furthermore, they establish testable criteria for investigating the prosthetic group structures of other green heme proteins by resonance Raman spectroscopy.     

Resonance raman studies of a c type algal cytochrome. Deuterium shifts and a comparison with mammalian cytochrome c.

A c type cytochrome isolated from Synechococcus lividus grown on water and 2H2O media, has been studied by resonance Raman spectroscopy. The spectra were taken on the oxidized and reduced protein with excitation within the Soret band at 441.6 nm to determine whether individual resonance Raman bands of the heme shift upon deuterium substitution and also to provide a comparison with the spectra of horse heart cytochrome c. Some of the shifts observed with the deuterated heme c are larger than the corresponding shifts in meso-deuterated metalloporphyrins suggesting mixing of peripheral substituent vibrations with the skeletal modes of the porphyrin macrocycle. The algal cytochrome exhibits resonance Raman spectra roughly similar to those of horse heart cytochrome c, consistent with its optical absorption spectra which is typical of c type cytochromes, although a detailed comparison reveals note-worthy differences between the spectra of the two proteins; this may be a reflection of the effect of non-methionine ligands and protein environment on the vibrations of the c type heme in the algal cytochrome.     

Understanding amyloid fibril formation using protein fragments: structural investigations via vibrational spectroscopy and solid-state NMR

It is well established that amyloid proteins play a primary role in neurodegenerative diseases. Alzheimer’s, Parkinson’s, type II diabetes, and Creutzfeldt-Jakob’s diseases are part of a wider family encompassing more than 50 human pathologies related to aggregation of proteins. Although this field of research is thoroughly investigated, several aspects of fibrillization remain misunderstood, which in turn slows down, or even impedes, advances in treating and curing amyloidoses. To solve this problem, several research groups have chosen to focus on short fragments of amyloid proteins, sequences that have been found to be of great importance for the amyloid formation process. Studying short peptides allows bypassing the complexity of working with full-length proteins and may provide important information relative to critical segments of amyloid proteins. To this end, efficient biophysical tools are required. In this review, we focus on two essential types of spectroscopic techniques, i.e., vibrational spectroscopy and its derivatives (conventional Raman scattering, deep-UV resonance Raman (DUVRR), Raman optical activity (ROA), surface-enhanced Raman spectroscopy (SERS), tip-enhanced Raman spectroscopy (TERS), infrared (IR) absorption spectroscopy, vibrational circular dichroism (VCD)) and solid-state nuclear magnetic resonance (ssNMR). These techniques revealed powerful to provide a better atomic and molecular comprehension of the amyloidogenic process and fibril structure. This review aims at underlining the information that these techniques can provide and at highlighting their strengths and weaknesses when studying amyloid fragments. Meaningful examples from the literature are provided for each technique, and their complementarity is stressed for the kinetic and structural characterization of amyloid fibril formation.     

大白鼠糖致白内障的激光喇曼光谱研究

作者用激光喇曼光谱法分析半乳糖导致大白鼠晶状体混浊过程中构象的变化。通过SPEX 1403型激光喇曼光谱仪得到了正常及不同混浊度晶状体的喇曼光谱。结果表明晶状体可溶性蛋白质二级结构的光谱未见异常,其残基酪氨酸及色氨酸微环境起了变化。随着晶状体混浊度的增加,SH谱峰强度变小而S-S键谱峰增强,同时观察到荧光背景逐渐加强。经分析认为晶状体混浊是与蛋白质分子的聚集有关。     

Vibrational Raman optical activity of proteins,nucleic acids,and viruses

Due to its sensitivity to chirality, Raman optical activity (ROA), which may be measured as a small difference in vibrational Raman scattering from chiral molecules in right- and left-circularly polarized incident light, is a powerful probe of biomolecular structure in solution. Protein ROA spectra provide information on the secondary and tertiary structures of the polypeptide backbone, hydration, side-chain conformation, and structural elements present in denatured states. Nucleic acid ROA spectra yield information on the sugar ring conformation, the base stacking arrangement, and the mutual orientation of the sugar and base rings around the C-N glycosidic linkage. ROA is able to simultaneously probe the structures of both the protein and the nucleic acid components of intact viruses. This article gives a brief account of the theory and measurement of ROA and presents the ROA spectra of a selection of proteins, nucleic acids, and viruses which illustrate the applications of ROA spectroscopy in biomolecular research.     

Raman structural markers of tryptophan and histidine side chains in proteins

The Raman spectrum of a protein contains a wealth of information on the structure and interaction of the protein. To extract the structural information from the Raman spectrum, it is necessary to identify and interpret the marker bands that reflect the structure and interaction in the protein. Recently, new Raman structural markers have been proposed for the tryptophan and histidine side chains by examining the spectra-structure correlations of model compounds. Raman structural markers are now available for the conformation, hydrogen bonding, hydrophobic interaction, and cation-pi interaction of the indole ring of Trp. For His, protonation, tautomerism, and metal coordination of the imidazole ring can be studied by using Raman markers. The high-resolution X-ray crystal structures of proteins provide the basis for testing and modifying the Raman structural markers of Trp and His. The structures derived from Raman spectra are generally consistent with the X-ray crystal structures, giving support for the applicability of most Raman structural makers. Possible modifications and limitations to some marker bands are also discussed.     

Average orientation of aromatic residues in proteins determined from linear dichroism spectroscopy. A comparison of results on bovine gamma-crystallins with X-ray data

The structures of the two very closely related proteins, bovine gamma II- and gamma IVa-crystallin have been studied by means of near-ultra-violet linear dichroism spectroscopy on squeezed polyacrylamide gel systems. The crystallin spectra are discussed in terms of the spectra of the aromatic chromophores present in these proteins and provide detailed information on the average orientation of these residues in the proteins. A comparison of our results with information based on crystallographic X-ray experiments shows excellent agreement, reflecting even some of the minor differences between the two proteins studied. Since linear dichroism measurements as performed here take a few days only, and can be done on most aqueous protein solutions, linear dichroism spectroscopy may give a valuable contribution to structural studies on proteins.     

Raman optical activity of proteins, carbohydrates and glycoproteins

On account of its sensitivity to chirality, Raman optical activity (ROA), which may be measured as a small difference in the intensity of vibrational Raman scattering from chiral molecules in right- and left-circularly polarized incident light, or as the intensity of a small circularly polarized component in the scattered light, is a powerful probe of the structure of biomolecules. Protein ROA spectra provide information on secondary and tertiary structures of polypeptide backbones, backbone hydration and side-chain conformations, and on structural elements present in unfolded states. Carbohydrate ROA spectra provide information on the central features of carbohydrate stereochemistry, especially that of the glycosidic link. Glycoprotein ROA spectra provide information on both the polypeptide and carbohydrate components. This article describes the ROA technique and presents and discusses the ROA spectra of a selection of proteins, carbohydrates, and a glycoprotein. The many structure-sensitive bands in protein ROA spectra are favorable for applying pattern recognition techniques, illustrated here using nonlinear mapping, to determine structural relationships between different proteins.     

The analysis of merino wool cuticle and cortical cells by Fourier transform Raman spectroscopy

Wool fibers are comprised of proteins known as α-keratins and have a complex morphological structure. The major components of this structure, the cuticle and cortical cells, differ in the conformations of their peptide chains as well as their amino acid compositions. High quality Fourier transform Raman spectra of cortical and cuticle cells isolated from fine Merino wool fibers have been obtained. Raman spectroscopy has been shown to be sensitive to the differences in both secondary structure and amino acid composition. The cortical cells were found to be higher in α-helical content as compared to the cuticle cells, which had an increased disordered content. Specific information, consistent with amino acid analysis results, regarding cystine, tyrosine, tryptophan, and phenylalanine residues, were obtained for both the cortical and cuticle cells. In addition, the Raman spectra provided information about free thiol groups, amino acids residues with amide group side chains, and residues with protonated carboxyl group side chains. Middle ir transmission spectra of these isolated cells were also obtained. In comparison to the Raman data, the middle ir spectra were found to be not as rich in information. © 1997 John Wiley & Sons, Inc. Biopoly 42: 7–17, 1997     

Vibrational spectra and structure of myelin membranes

Raman and infrared spectroscopy have been simultaneously applied, for the first time, to the study of myelin membranes and their proteolipid protein (PLP) so as to obtain information on the secondary structure of proteins and the ordering of lipid chains. The vibrational spectra were recorded at physiological pH using a non-denaturing detergent (n-octyl--d-glucopyranoside) in phosphate buffer. Neither the buffer nor the detergent interfere spectroscopically with the amide bands from proteins. The spectra reveal that the predominant secondary structure in the polypeptide backbone in myelin is the helix. The proteolipid protein was found to be more disordered than the polypeptide arrangement of the myelin membrane, as deduced from the relative intensities and halfwidths of characteristic infrared amide I bands. -form and turns are also present, the amount of these structures being higher in PLP. The study of the Raman spectra of vC-C and vC-H regions made it possible to obtain information on the lipid chain order.     

Spectroscopic characterization of (57)Fe-enriched cytochrome c

Investigation of the heme iron dynamics in cytochrome c with Mössbauer spectroscopy and especially nuclear resonance vibrational spectroscopy requires the replacement of the natural abundant heme iron with the ⁵⁷Fe isotope. For demetallization, we use a safer and milder ferrous sulfate–hydrochloric acid method in addition to the harsher commonly used hydrofluoric acid-based procedure. The structural integrity of the ⁵⁷Fe-reconstituted protein in both oxidation states is confirmed from absorption spectra and a detailed analysis of the rich resonance Raman spectra. These results reinforce the application of metal-substituted heme c proteins as reliable models for the native proteins.     

Single seed Raman measurements allow taxonomical discrimination of Apiaceae accessions collected in gene banks

NIR-FT-Raman spectroscopy was applied for a nondestructive analysis of single seeds (fruit mericarps) of 36 accessions belonging to various species of the Apiaceae family. Main seed components such as fatty acids, polysaccharides, proteins, and lignin were identified based on the obtained Raman spectra. Variation at the species and genus level was related to differences observed between spectra. The application of cluster analysis discriminated among most of the species evaluated and grouped them according to their taxonomical classification. The spectroscopically analyzed seeds germinated and developed into normal seedlings to demonstrate the additional advantage that Raman spectroscopy is nondestructive and can be applied to living seed without harm. These results indicate that Raman spectroscopy is a valuable tool for the rational evaluation and management of genetic resources in ex situ seed collections by providing useful information for taxonomical validation of the accessions.     

Resonance Raman spectroscopy of methylamine dehydrogenase from bacterium W3A1

Resonance Raman spectroscopy has been used to probe the structure of the covalently bound quinone cofactor in methylamine dehydrogenase from the bacterium W3A1. Spectra were obtained on the phenylhydrazine and 2-pyridylhydrazine derivatives of the native enzyme, on the quinone-containing subunit labeled with phenylhydrazine, and on an active-site peptide also labeled with phenylhydrazine. Comparisons of these spectra to the corresponding spectra of copper-containing amine oxidase derivatives indicate that the quinones in these two classes of quinoproteins are not identical. The resonance Raman spectra of the native enzyme and small subunit have also been measured. 16O/18O exchange permitted the carbonyl modes of the quinone to be identified in the resonance Raman spectrum of oxidized methylamine dehydrogenase: a band at 1614 cm-1, together with a shoulder at 1630 cm-1, are assigned as modes containing substantial C = O stretching character. D2O/H2O exchange has pronounced effects on the resonance Raman spectrum of the oxidized enzyme, suggesting that the quinone may have numerous hydrogen bonds to the protein or that it is unusually sensitive to the local environment. Resonance Raman spectra of the isolated small subunit, and its phenylhydrazine derivative, are considerably different from the corresponding spectra of the intact protein. An attractive explanation for these observations is that the quinone cofactor in methylamine dehydrogenase from W3A1 is located at the interface between the large and small subunits, as found for the enzyme from Thiobacillus versutus [Vellieux, F. M. D., Huitema, F., Groendijk, H., Kalk, K. H., Frank, J. Jzn., Jongejan, J. A., & Duine, J. A. (1989) EMBO J. 8, 2171-2178].(ABSTRACT TRUNCATED AT 250 WORDS)     

Surface-enhanced resonance raman scattering spectroscopy of bacterial photosynthetic membranes: orientation of the carotenoids of Rhodobacter sphaeroides 2.4.1

Surface-enhanced resonance Raman scattering (SERRS) spectra were obtained from carotenoids, in the all-trans configuration, located on the antenna complexes of Rhodobacter sphaeroides 2.4.1 membranes. Since resonance Raman (RR) spectra are barely detectable at the concentration that SERRS signals saturate, SERRS represents a very sensitive means of detecting pigments in biological systems. Prominent SERRS spectra of sphaeroidenone were detected in chromatophores (cytoplasmic side out) but not in spheroplast-derived vesicles (periplasmic side out), demonstrating that the carotenoid is asymmetrically located on the cytoplasmic side of the cell membrane. Comparison of peak frequencies from SERRS and RR spectral data suggests that the carotenoids are oriented into the membrane with the methoxy end of the isoprenoid chains located closest to the cytoplasmic side of the intracytoplasmic membrane. This work not only shows that SERRS spectroscopy can provide information on the location of a chromophore in a biological membrane but also for the first time demonstrates that SERRS data can be used to ascertain the orientation of a chromophore within the membrane. This observation greatly increases the potential of this technique for structural analysis of intact membranes at the molecular level.     

Phage phi X174 probed by laser Raman spectroscopy: evidence for capsid-imposed constraint on DNA secondary structure

The Raman spectrum of the isometric bacteriophage phi X174 contains a number of well-resolved bands which have been assigned unambiguously to proteins of the capsid or to the single-stranded DNA (ssDNA) genome. Additional Raman bands of protein and DNA, which are partially overlapped in the spectrum of virus, have been resolution enhanced by Fourier deconvolution to permit improved semiquantitative measurement of spectral intensities and frequencies for structural conclusions. Raman conformation markers indicate that the ssDNA molecule within the capsid contains nucleosides of C2'-endo sugar pucker and anti-glycoside bond orientation, but the nucleic acid backbone lacks the geometry characteristic of B-form DNA. The Raman profile of encapsidated phi X DNA indicates a backbone more similar to heat-denatured DNA than to DNA containing hairpinlike secondary structure. This finding suggests limited interbase interactions in the packaged genome, which is presumably the result of constraints imposed by the viral capsid. Thus, the extensive pairing and stacking of bases indicated by Raman profiles from ssRNA viruses are not evident for the phi X174 chromosome. Overall, the proteins of the virion contain extensive beta-sheet and irregular secondary structures. Fourier deconvolution of the Raman amide I band provides an estimate of the percentage of total beta-sheet structure (approximately 60%) in all proteins of the virion. The amide III region of the spectrum confirms that beta-sheet and irregular domains are the predominant protein secondary structures. Samples of phi X174 concentrated for Raman spectroscopy by either ultracentrifugation or ultrafiltration exhibit nearly identical Raman spectra, indicating that either method can be employed to prepare intact virus without significant loss of DNA or protein components.     

Exclusively NOESY-based automated NMR assignment and structure determination of proteins

A fully automated method is presented for determining NMR solution structures of proteins using exclusively NOESY spectra as input, obviating the need to measure any spectra only for obtaining resonance assignments but devoid of structural information. Applied to two small proteins, the approach yielded structures that coincided closely with conventionally determined structures.