Structural properties of bombesin-like peptides revealed by surface-enhanced Raman scattering on roughened silver electrodes

This work presents a Fourier-transform absorption infrared, Fourier-transform Raman, and surface-enhanced Raman scattering (SERS) study of the following peptides belonging to the bombesin-like family: phyllolitorin, [Leu(8)]phyllolitorin, NMB, NMC, and PG-L. The SERS study was undertaken to understand the adsorption mechanism of bombesin-like peptides on an electrochemically roughened silver electrode surface and to show changes in the adsorption mechanism with alterations in amino acids and small tertiary structures. The SERS spectra presented here shows bands mainly associated with the Trp(8) residue vibrations. The presence of mainly pyrrole coring vibrations for phyllolitorin and [Leu(8)]phyllolitorin and mainly benzene coring modes for NMB and NMC indicated that these groups interact with the roughened silver electrode surface. Furthermore, N(1)--C(8) and C(3)--C(9) bonds of the PG-L indole ring seemed to have nearly a vertical orientation on the electrode surface. In addition, distinct vibrations of the C--S fragment were observed in the SERS spectra of [Leu(8)]phyllolitorin and PG-L. The strong enhancement of the nu(C==O) vibration in the [Leu(8)]phyllolitorin SERS spectrum yielded evidence that the intact C==O bond(s) bind strongly to the silver electrode surface, whereas NMC, phyllolitorin, and NMB were located near the silver surface. This finding was supported by the presence of the nu(C--C(==O)) mode. The amide I band observed at 1642 and 1634 cm(-1) for NMB and NMC, respectively, and the Raman amide III band seen in the 1282-1249 cm(-1) range for all peptides except PG-L, indicate that the strongly hydrogen-bonded alpha-helical conformation and random-coil structure are favored for binding to the surface. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 980-992, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Raman spectroscopy and order in biological systems

The Raman spectra in the low 5–200 cm⁻¹ frequency region of metabolically activeE. coli cells have been analyzed to determine whether they are indicators of a possible in vivo underlying order by applying standard concepts derived from the Raman spectroscopy of crystalline systems with varying degrees of order. The analysis suggests that in-vivo space-time ordered structures involving amino acids associated with DNA exist since the low frequency lines of metabolically active cells can be assigned to lines seen in the spectra of crystals of given amino acids known to associated with DNA early in the lifetime of a cell.     

Subpicosecond dynamics in nucleotides measured by spontaneous Raman spectroscopy

The band widths in Raman spectra are sensitive to dynamics active on a time scale from 0.1 to 10 ps. The band widths of nucleotide vibrations and their dependence on temperature, concentration, and structure are reported. From the experimental band widths and second moments, it is derived that the adenine vibrations at 725, 1336, 1480, and 1575 cm⁻¹, and the uracil vibration at 787 cm⁻¹, are in the fast modulation limit. The correlation times of the perturbations are faster than 0.4 ps. Thermal melting of the helical structure in polynucleotides results in larger band widths, due to an increase in vibrational dephasing and energy relaxation as a consequence of the increased interaction of the base moieties with the solvent molecules. The band width of the 725 cm⁻¹ adenine vibration is dependent on the type and structure of the backbone. It is found to be perturbed by movements of the sugar-phosphate moiety relative to the base. The band width of the 1575 cm⁻¹ adenine vibration is found to be sensitive to the base-pairing interaction. From a comparison of the band widths in polynucleotides with a different base sequence (homopolymer vs alternating purine-pyrimidine sequence), it is concluded that resonant vibrational energy transfer between the base molecules is not important as a relaxation process for the vibrational band widths of nucleotides. Several theoretical models for the interpretation of band widths are discussed. The theory does not take into account the strong hydrogen-bonding nature water and hence fails to describe the observations in nucleotide-water systems. The bands of the carbonyl stretching vibrations are inhomogeneously broadened. The carbonyl groups have a strong dipolar interaction with the polar water molecules and are therefore strongly perturbed by coupling to the heatbath via hydrogen bonds. © 1997 John Wiley & Sons, Inc. Biopoly 41: 751–763, 1997     

拉曼光谱检测微生物的研究方法和进展北大核心

快速准确地识别和鉴定微生物对于环境科、食品质量以及医学诊断等领域研究至关重要。拉曼光谱(Raman spectroscopy)已经被证明是一种能够实现微生物快速诊断的新技术,在提供微生物指纹图谱信息的同时,能够快速、非标记、无创、敏感地在固体和液体环境中实现微生物单细胞水平的检测。本文简单介绍了拉曼光谱的基本概念和原理,重点综述了拉曼光谱微生物检测应用中的样品处理方法及光谱数据处理方法。除此之外,本文概括了拉曼光谱在细菌、病毒和真菌中的应用,其中单独概括了拉曼在细菌快速鉴定和抗生素药敏检测中的应用。最后,本文阐述了拉曼光谱在微生物检测中的挑战和展望。     

Structural analysis of biological aliphatic compounds using surface-enhanced Fourier transform Raman spectroscopy

The surface-enhanced Raman scattering (SERS) technique for Fourier transform Raman spectrometry is employed to reveal the chemical structure of biological aliphatic compounds consisting of folded, long aliphatic chains. The structural analysis is performed via the measurements of the accordion-vibration modes generated in the ordered, long aliphatic chain. The SERS spectra after subtraction of a background spectrum give segment lengths that are almost perfectly consistent with the chemical structures studied by mass spectrometry. The agreement of the SERS results with those of mass spectrometry suggests the positions of kinks in the long hydrocarbon chain. The combination technique of SERS and mass spectrometry is useful to discuss the structure of folded, long biological lipids.     

Structural analysis of arabinoxylans isolated from native and malted finger millet (Eleusine coracana, ragi)

Structural elucidation of purified arabinoxylans isolated from finger millet and its malt by methylation, GLC-MS, periodate oxidation, Smith degradation, NMR, IR, optical rotation, and oligosaccharide analysis indicated that the backbone was a 1,4-beta-D-xylan, with the majority of the residues substituted at C-3. The major oligosaccharide generated by endo xylanase treatment was homogeneous with a molecular weight of 1865 Da corresponding to 14 pentose residues as determined by MALDI-TOF-MS and gel filtration on Biogel P-2. The structural analysis of this oligosaccharide showed that it contained 8 xylose and 6 arabinose residues, substituted at C-3 (monosubstituted) and at both C-2 and C-3 (disubstituted).     

Bladder tissue characterization using probe‐based Raman spectroscopy: Evaluation of tissue heterogeneity and influence on the model prediction

Existing approaches for early‐stage bladder tumor diagnosis largely depend on invasive and time‐consuming procedures, resulting in hospitalization, bleeding, bladder perforation, infection and other health risks for the patient. The reduction of current risk factors, while maintaining or even improving the diagnostic precision, is an underlying factor in clinical instrumentation research. For example, for clinic surveillance of patients with a history of noninvasive bladder tumors real‐time tumor diagnosis can enable immediate laser‐based removal of tumors using flexible cystoscopes in the outpatient clinic. Therefore, novel diagnostic modalities are required that can provide real‐time in vivo tumor diagnosis. Raman spectroscopy provides biochemical information of tissue samples ex vivo and in vivo and without the need for complicated sample preparation and staining procedures. For the past decade there has been a rise in applications to diagnose and characterize early cancer in different organs, such as in head and neck, colon and stomach, but also different pathologies, for example, inflammation and atherosclerotic plaques. Bladder pathology has also been studied but only with little attention to aspects that can influence the diagnosis, such as tissue heterogeneity, data preprocessing and model development. The present study presents a clinical investigative study on bladder biopsies to characterize the tumor grading ex vivo, using a compact fiber probe‐based imaging Raman system, as a crucial step towards in vivo Raman endoscopy. Furthermore, this study presents an evaluation of the tissue heterogeneity of highly fluorescent bladder tissues, and the multivariate statistical analysis for discrimination between nontumor tissue, and low‐ and high‐grade tumor.     

Probing the cell wall heterogeneity of micro-dissected wheat caryopsis using both active and inactive forms of a GH11 xylanase

The external envelope of wheat grain (Triticum aestivum L. cv. Isengrain) is a natural composite whose tissular and cellular heterogeneity constitute a significant barrier for enzymatic cell wall disassembly. To better understand the way in which the cell wall network and tissular organization hamper enzyme penetration, we have devised a strategy based on in situ visualization of an active and an inactive form of a xylanase in whole-wheat bran and in three micro-dissected layers (the outer bran, the inner bran and the aleurone layer). The main aims of this study were to (1) evaluate the role of cuticular layers as obstacles to enzyme diffusion, (2) assess the impact of the cell wall network on xylanase penetration, (3) highlight wall heterogeneity. To conduct this study, we created by in vitro mutagenesis a hydrolytically inactive xylanase that displayed full substrate binding ability, as demonstrated by the calculation of dissociation constants (K_d) using fluorescence titration. To examine enzyme penetration and action, immunocytochemical localization of the xylanases and of feebly substituted arabinoxylans (AXs) was performed following incubation of the bran layers, or whole bran with active and inactive isoforms of the enzyme for different time periods. The data obtained showed that the micro-dissected layers provided an increased accessible surface for the xylanase and that the enzyme-targeted cell walls were penetrated more quickly than those in intact bran. Examination of immunolabelling of xylanase indicated that the cuticle layers constitute a barrier for enzyme penetration in bran. Moreover, our data indicated that the cell wall network by itself physically restricts enzyme penetration. Inactive xylanase penetration was much lower than that of the active form, whose penetration was facilitated by the concomitant depletion of AXs in enzyme-sensitive cell walls.     

Discrimination of normal, benign, and malignant breast tissues by Raman spectroscopy

Breast cancers are the leading cancers among females. Diagnosis by fine needle aspiration cytology (FNAC) is the gold standard. The widely practiced screening method, mammography, suffers from high false positive results and repeated exposure to harmful ionizing radiation. As with all other cancers survival rates are shown to heavily depend on stage of the cancers (Stage 0, 95%; Stage IV, 75%). Hence development of more reliable screening and diagnosis methodology is of considerable interest in breast cancer management. Raman spectra of normal, benign, and malignant breast tissue show significant differences. Spectral differences between normal and diseased breast tissues are more pronounced than between the two pathological conditions, malignant and benign tissues. Based on spectral profiles, the presence of lipids (1078, 1267, 1301, 1440, 1654, 1746 cm(-1)) is indicated in normal tissue and proteins (stronger amide I, red shifted DeltaCH2, broad and strong amide III, 1002, 1033, 1530, 1556 cm(-1)) are found in benign and malignant tissues. The major differences between benign and malignant tissue spectra are malignant tissues seem to have an excess of lipids (1082, 1301, 1440 cm(-1)) and presence of excess proteins (amide I, amide III, red shifted DeltaCH2, 1033, 1002 cm(-1)) is indicated in benign spectra. The multivariate statistical tool, principal components analysis (PCA) is employed for developing discrimination methods. A score of factor 1 provided a reasonable classification of all three tissue types. The analysis is further fine-tuned by employing Mahalanobis distance and spectral residuals as discriminating parameters. This approach is tested both retrospectively and prospectively. The limit test, which provides the most unambiguous discrimination, is also considered and this approach clearly discriminated all three tissue types. These results further support the efficacy of Raman spectroscopic methods in discriminating normal and diseased breast tissues.     

The pH-dependent structure of calf thymus DNA studied by Raman spectroscopy

The pH-dependent structure of calf thymus DNA is analyzed using Raman spectroscopy. The Raman spectra in the acidic region demonstrate that denaturation occurs in several steps. The binding of H⁺ to adenine and cytosine residues is accompanied by a decrease in the percentage of DNA in the B-conformation and a concurrent increase in a conformation most probably related to the C-form. The denaturation of DNA is observed at pH 3.3 and parallels the protonation of guanine bases. The Raman spectra of calf thymus DNA in the basic region (above pH 10) show that guanine residues are deprotonated at a lower pH value than are thymine residues. In addition, Raman spectra in the basic region detect conformational changes of the phosphate backbone different from those found in the acidic region.     

Theoretical and pH dependent surface enhanced Raman spectroscopy study on caffeine

The surface enhanced Raman spectroscopy (SERS) spectrum of caffeine is recorded on a silver colloid at different pH values. It is discussed on the basis of the SERS "surface selection rules" in order to characterize its vibrational behavior on such a biological artificial model. To improve the previous assignments in the Raman spectrum and for a reliable, detailed analysis of SERS spectra, density functional theory calculations (structural parameters, harmonic vibrational wavenumbers, total electron density, and natural population analysis of the molecule) are performed for the anhydrous form of caffeine and the results are discussed. The predicted geometry and vibrational Raman spectra are in good agreement with the experimental data. The flat orientation of the mainly chemisorbed caffeine attached through the pi electrons and the lone pair of nonmethylated N atoms of the imidazole ring are proposed to occur at neutral and basic pH values. At acid pH values caffeine is probably adsorbed on the Ag surface through one or both oxygen atoms, more probably through the O atom of the conjugated carbonyl group with an end-on orientation. However, the changes in the overall SERS spectral pattern seem to indicate the electromagnetic mechanism as being the dominant one.     

Study of ATP binding in the active site of Na,K-ATPase as probed by ultraviolet resonance Raman spectroscopy

The ultraviolet resonance Raman (UV RR) spectra of functional ATP/membrane-bound Na⁺K⁺-ATPase complexes have been obtained. The substrate binding in the enzyme active site has been shown to be accompanied with significant changes in the electronic vibrational structure of the adenine ring. From the spectral analysis of ATP, 8-Br-ATP and 6-NHMe-adenine at various pH values the conclusion was made that N1 and the NH₂, group and, probably, N7 of the substrate adenine part, interact with the protein surroundings via hydrogen bonds.     

Resonance Raman spectroscopy in malaria research

In recent years, the field of Raman spectroscopy has witnessed a surge in technological development, with the incorporation of ultrasensitive, charge-coupled devices, improved laser sources and precision Rayleigh-filter systems. This has led to the development of sensitive confocal micro-Raman spectrometers and imaging spectrometers that are capable of obtaining high spatial-resolution spectra and images of subcellular components within single living cells. This review reports on the application of resonance micro-Raman spectroscopy to the study of malaria pigment (hemozoin), a by-product of hemoglobin catabolization by the malaria parasite, which is an important target site for antimalarial drugs. The review aims to briefly describe recent studies on the application of this technology, elucidate molecular and electronic properties of the malaria pigment and its synthetic analog β-hematin, provide insight into the mechanism of hemozoin formation within the food vacuole of the parasite, and comment on developing strategies for using this technology in drug-screening protocols.     

表面增强拉曼光谱技术在微生物鉴定中的应用进展

拉曼光谱自20世纪20年代被发现以来,经过近90年的发展,产生了许多分支。其中表面增强拉曼光谱是利用被测物质与粗糙金属表面的相互作用来提高拉曼光谱的信噪比,从而得到敏感度和精确度更高的图谱,可以将样品在不经过预处理的情况下对其进行快速检测。本文综述了表面增强拉曼光谱技术的原理、分类及鉴定特点,总结了该技术在食品、化学、医药、工业、病原等微生物学科的临床应用,进一步阐述了研究该技术的必要性和应用前景,旨在为从事该领域的科研人员提供参考依据。     

Studies of the retrogradation process for various starch gels using Raman spectroscopy

The retrogradation of untreated wild-type starches (potato, maize, and wheat), waxy maize starches, and one pregelatinized, modified amylose-rich starch was investigated continuously using Raman spectroscopy. The method detects conformational changes due to the multi-stage retrogradation, the rate of which differs between the starches. The pregelatinized, modified amylose-rich starch shows all stages of retrogradation in the course of its Raman spectra. In comparison to amylose, the retrogradation of amylopectin is faster at the beginning of the measurements and slower in the later stages. The untreated starches can be ranked in the order of their rate of retrogradation as follows: potato>maize>wheat.     

Active site heterogeneity in dimethyl sulfoxide reductase from Rhodobacter capsulatus revealed by Raman spectroscopy

Raman spectroscopy has been used to investigate the structure of the molybdenum cofactor in DMSO reductase from Rhodobacter capsulatus. Three oxidized forms of the enzyme, designated 'redox cycled', 'as prepared', and DMSOR(mod)D, have been studied using 752 nm laser excitation. In addition, two reduced forms of DMSO reductase, prepared either anaerobically using DMS or using dithionite, have been characterized. The 'redox cycled' form has a single band in the Mo=O stretching region at 865 cm(-1) consistent with other studies. This oxo ligand is found to be exchangeable directly with DMS(18)O or by redox cycling. Furthermore, deuteration experiments demonstrate that the oxo ligand in the oxidized enzyme has some hydroxo character, which is ascribed to a hydrogen bonding interaction with Trp 116. There is also evidence from the labeling studies for a modified dithiolene sulfur atom, which could be present as a sulfoxide. In addition to the 865 cm(-1) band, an extra band at 818 cm(-1) is observed in the Mo=O stretching region of the 'as prepared' enzyme which is not present in the 'redox cycled' enzyme. Based on the spectra of unlabeled and labeled DMS reduced enzyme, the band at 818 cm(-1) is assigned to the S=O stretch of a coordinated DMSO molecule. The DMSOR(mod)D form, identified by its characteristic Raman spectrum, is also present in the 'as prepared' enzyme preparation but not after redox cycling. The complex mixture of forms identified in the 'as prepared' enzyme reveals a substantial degree of active site heterogeneity in DMSO reductase.     

Structural changes in cartilage and collagen studied by high temperature Raman spectroscopy

Understanding the high temperature behavior of collagen and collagenous tissue is important for surgical procedures and biomaterials processing for the food, pharmaceutical, and cosmetics industries. One primary event for proteins is thermal denaturation that involves unfolding the polypeptide chains while maintaining the primary structure intact. Collagen in the extracellular matrix of cartilage and other connective tissue is a hierarchical material containing bundles of triple‐helical fibers associated with water and proteoglycan components. Thermal analysis of dehydrated collagen indicates irreversible denaturation at high temperature between 135°C and 200°C, with another reversible event at ～60‐80°C for hydrated samples. We report high temperature Raman spectra for freeze‐dried cartilage samples that show an increase in laser‐excited fluorescence interpreted as conformational changes associated with denaturation above 140°C. Spectra for separated collagen and proteoglycan fractions extracted from cartilage indicate the changes are associated with collagen. The Raman data also show appearance of new features indicating peptide bond hydrolysis at high temperature implying that molecular H₂O is retained within the freeze‐dried tissue. This is confirmed by thermogravimetric analysis that show 5‐7 wt% H₂O remaining within freeze‐dried cartilage that is released progressively upon heating up to 200°C. Spectra obtained after exposure to high temperature and re‐hydration following recovery indicate that the capacity of the denatured collagen to re‐absorb water is reduced. Our results are important for revealing the presence of bound H₂O within the collagen component of connective tissue even after freeze‐drying and its role in denaturation that is accompanied by or perhaps preceded by breakdown of the primary polypeptide structure.     

The molecular interaction of a protein in highly concentrated solution investigated by Raman spectroscopy

We used Raman spectroscopy to investigate the structure and interactions of lysozyme molecules in solution over a wide range of concentrations (2.5–300 mg ml^?1). No changes in the amide‐I band were observed as the concentration was increased, but the width of the Trp band at 1555 cm^?1 and the ratios of the intensities of the Tyr bands at 856 and 837 cm^?1, the Trp bands at 870 and 877 cm^?1, and the bands at 2940 (CH stretching) and 3420 cm^?1 (OH stretching) changed as the concentration was changed. These results reveal that although the distance between lysozyme molecules changed by more than an order of magnitude over the tested concentration range, the secondary structure of the protein did not change. The changes in the molecular interactions occurred in a stepwise process as the order of magnitude of the distance between molecules changed. These results suggest that Raman bands can be used as markers to investigate the behavior of high‐concentration solutions of proteins and that the use of Raman spectroscopy will lead to progress in our understanding not only of the basic science of protein behavior under concentrated (i.e., crowded) conditions but also of practical processes involving proteins, such as in the field of biopharmaceuticals. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 237–246, 2015.     

Structural alterations of the heme environment of cytochrome P450cam and the Y96F mutant as deduced by resonance Raman spectroscopy

Resonance Raman spectroscopy at 2.5cm(-1) resolution was used to probe differences in wild-type and Y96F mutant P450cam (CYP101), both with and without bound camphor or styrene substrates. In the substrate-free state, the spin state equilibrium is shifted from 6-coordinate low spin (6CLS) toward more 5-coordinate high spin (5CHS) when tyrosine-96 in the substrate pocket is replaced by phenylalanine. About 25% of substrate-free Y96F mutant is 5CHS as opposed to 8% for substrate-free wild-type P450cam. Spin equilibrium constants calculated from Raman intensities indicate that the driving force for electron transfer from putidaredoxin, the natural redox partner of P450cam, is significantly smaller on styrene binding than for camphor binding. Spectral differences suggest that there is a tilt in camphor toward the pyrrole III ring on Y96F mutation. This finding is consistent with the altered product distribution found for camphor hydroxylation by the Y96F mutant relative to the single enantiomer produced by the wild-type enzyme.     

Dispersive Raman spectroscopy allows the identification and quantification of melanin types

Melanins are the most prevalent pigments in animals and are involved in visual communication by producing colored traits that often evolve as intraspecific signals of quality. Identifying and quantifying melanins are therefore essential to understand the function and evolution of melanin‐based signals. However, the analysis of melanins is difficult due to their insolubility and the lack of simple methods that allow the identification of their chemical forms. We recently proposed the use of Raman spectroscopy as a simple, noninvasive technique that can be used to identify and quantify melanins in feathers and hairs. Contrarily, other authors later stated that melanins are characterized by a lack of defined Raman signals. Here, we use confocal Raman microscopy to confirm previous analyses showing that the two main chemical forms of melanins (eumelanin and pheomelanin) exhibit distinct Raman signal and compare different excitation wavelengths to analyze synthetic pheomelanin and natural melanins in feathers of different species of birds. Our analyses indicate that only laser excitation wavelengths below 1064 nm are useful for the analysis of melanins by Raman spectroscopy, and only 780‐nm laser in the case of melanins in feathers. These findings show that the capacity of Raman spectroscopy to distinguish different chemical forms of melanins depends on laser power and integration time. As a consequence, Raman spectroscopy should be applied after preliminar analyses using a range of these parameters, especially in fragile biological tissues such as feathers.