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。     

喇曼光谱技术在生物医学中的应用

喇曼光谱技术是一种非侵入、非弹性散射技术,能够在分子层次上探测物质的临床医学特征和结构特征。本文综述了近十年来喇曼光谱技术在生物医学中的最新发展,归纳出了四种目前在生物医学中最为活跃的喇曼光谱技术：近红外喇曼光谱、紫外共振喇曼光谱、表面增强喇曼光谱和多维喇曼成像技术。详细阐述了这四种技术的特点和适用范围,并且列举了丰富的成功范例。     

共振喇曼光谱技术在恶性肿瘤诊断中的应用

目的：探讨共振喇曼光谱技术用于早期恶性肿瘤诊断的研究。方法：利用氩离子激光作为线偏振光的特点,采集偏振荧光光谱,对荧光光谱的偏振态进行分析。利用不同荧光物质的荧光可能具有不同偏振态的特点减少其它荧光物质的荧光对光谱分析的影响。血清样品产生的荧光也具有确定的偏振性。对所检测病人血清经激光分析仪进行喇曼光谱技术分析,光谱数据经计算机软件处理,自动显示图谱和数据,并直接给出各项指标及诊断提示。本结果与细胞病理学结果进行了对照研究。结果：恶性肿瘤样本176例,检测出阳性病例141例,阳性符合率为80.1%;良性肿瘤样本52例,4例阳性,假阳性率为7.7%;正常体检样本248例,检测结果均为阴性。结论：喇曼光谱技术适用于肿瘤初筛、普查及早期诊断,有推广应用前途。     

共振喇曼光谱技术在恶性肿瘤诊断中的应用

目的:探讨共振喇曼光谱技术用于早期恶性肿瘤诊断的研究。方法:利用氩离子激光作为线偏振光的特点,采集偏振荧光光谱,对荧光光谱的偏振态进行分析。利用不同荧光物质的荧光可能具有不同偏振态的特点减少其它荧光物质的荧光对光谱分析的影响。血清样品产生的荧光也具有确定的偏振性。对所检测病人血清经激光分析仪进行喇曼光谱技术分析,光谱数据经计算机软件处理,自动显示图谱和数据,并直接给出各项指标及诊断提示。本结果与细胞病理学结果进行了对照研究。结果:恶性肿瘤样本176例,检测出阳性病例141例,阳性符合率为80.1%;良性肿瘤样本52例,4例阳性,假阳性率为7.7%;正常体检样本248例,检测结果均为阴性。结论:喇曼光谱技术适用于肿瘤初筛、普查及早期诊断,有推广应用前途。     

水稻病毒激光喇曼光谱的初步探讨

本文初步探讨水稻病毒激光喇曼光谱,分析了谱线与病毒中核酸蛋白质结构的关系。确信应用激光喇曼光谱研究水稻病毒是具有信息量大、高灵敏及高分辨本领的。     

激光喇曼光谱技术在食品科学中的应用

激光喇曼光谱技术是一种非侵入、非弹性的光散射技术,它能够无损地提供丰富的分子结构和物质成分的信息。近来它在食品工业领域表现出很大的应用潜力。本文综述了激光喇曼光谱技术在食品科学中的应用及其新进展。主要包括果蔬农药残留的检测、肉类产品质量检测、伪劣食品鉴定、食物蛋白的研究以及食品加工监控等方面的应用。并对喇曼光谱技术在这些方面的应用前景作了进一步的展望。     

寡聚脱氧核苷酸d(G-C)_3的激光喇曼谱及其分析

用改进的固相磷酰三酯法合成了oligo-d(G-C)_3。以氩离子激光为激发光源,波长488nm.,在室温条件下,分别测定了纯化后的oligo-d(G-C)_3和其组分单体5’-dGMP和5’-dCMP的激光喇曼谱。观察到被测定的物质在300-2500cm~(-1)频率区间,各自都有其特征的谱形和喇曼峰。5’-dGMP和5’-dCMP谱中大多数特征峰在寡聚体的谱中消失,而在oligo-d(G-C)_3谱中出现了几处新的喇曼峰。经查证,峰832,851和899cm~(-1)系糖-磷酸主链的特征喇曼峰,另外几处峰与DNA的构象有关。实验结果表明oligo-d(G-C)_3在水溶液中(室温)主要以B-构象存在。     

用共振喇曼光谱研究血卟啉衍生物对紫膜菌紫质的光敏作用

本文用准共振喇曼光谱（ＲＲＳ）作为研究光动力损伤过程的探针,从分子水平上研究了光敏剂血卟啉衍生物与紫膜菌紫质的作用。在观察到损伤现象的基础上对损伤部位进行了定位。初步确定生色团视黄醛中Ｃ－Ｃ和Ｃ＝Ｃ等键为其易受损部位。     

用共振喇曼光谱研究血卟啉衍生笺对紫膜菌紫质的光敏作用

本文用准共振喇曼光谱作为研究光动力损伤过程的探讨，从分子水平上研究了光敏剂血卟啉衍生物与紫膜菌紫质的作用。在观察到损伤现象的基础上对损伤部位进行了定位。初步确定生色团视黄醛中Ｃ－Ｃ和Ｃ＝Ｃ等键为其易受损部位。     

干旱胁迫对黄瓜PSⅡ颗粒铁组分Mssbauer谱的影响(英文)

黄瓜 (CucumissativusL .)叶片PSⅡ颗粒的M ssbauer谱呈现 4套双峰 ,依它们的化学位移和四极矩劈裂数值 ,分别属于氧化态Cyt_b5 5 9、还原态Cyt_b5 5 9、Fe3 _Q复合物和Fe2 _Q复合物。干旱胁迫影响QA/QB 中铁 (Fe)参与电子传递的速率 ,使PSⅡ颗粒的M ssbauer谱中Fe2 的吸收双峰消失 ,即还原态Cyt_b5 5 9转变为氧化态Cyt_b5 5 9,Fe2 _Q复合物转变为Fe3 _Q复合物。结果干旱胁迫改变了铁醌复合物中的铁存在状态 ,使铁具有氧化_还原态变化 ,从而参与电子传递。     

Structural analysis and comparison of cobrotoxin and cardiotoxins by near-IR Fourier transform Raman spectroscopy.

Venom toxins were isolated from Formosan cobra (Naja naja atra) by cation-exchange chromatography. The near-IR FT-Raman analytical method has been applied to the characterization and classification of the toxin components in their lyophilized forms. Structural analysis and comparison of various purified toxin fractions were made with respect to their amino acid compositions and near-IR Fourier-transform Raman spectra. The results indicate that the major secondary structure of cobra toxins including cobrotoxin and various cardiotoxins is mainly anti-parallel beta-pleated sheet as judged by the Raman signals at 1238 cm-1 (amide III) and 1671 cm-1 (amide I). It is also found that the relative Raman signal intensities of Tyr, Phe, Trp and Met residues in purified toxins correlate very well with the structural data obtained from amino acid analysis. The advantage and improvement of applying the near-IR FT-Raman spectroscopy to the unambiguous classification and comparison of venom toxins are evident and the discrepancies with previous Raman studies on these venom toxins are also revealed and discussed.     

Near-infrared Fourier transform Raman and conventional Raman studies of calf gamma-crystallins in the lyophilized state and in solution.

We present in this report a detailed structural study of calf gamma-crystallins both in the solid state and in solution by the newly developed technique of near-infrared (IR) Fourier transform (FT)-Raman spectroscopy as well as by the conventional Raman method. In comparison with conventional laser Raman spectroscopy, the near-IR FT-Raman approach exhibits several attractive features such as fluorescence rejection capability, frequency accuracy, and the FT's multiplex and throughput advantages. These distinct characteristics combined form the basis for the particular suitability of FT-Raman in crystallin structural analysis and elucidation. We have thus obtained evidence in support of the view that native calf gamma-II crystallin does not contain a disulfide bond either in the lyophilized state or in solution. In addition, conventional Raman spectra are examined for all four gamma-crystallin fractions. gamma-S, gamma-II, gamma-III, and gamma-IV, and the results indicate a high degree of structural similarities among them. It is also found that the sulfhydryl groups in all four gamma-crystallins are highly resistant to air oxidation and are capable of maintaining their reduced state during isolation in the absence of added reductants or such chelating agents as EDTA.     

An anomaly in the resonance Raman spectra of cytochrome P-450cam in the ferrous high-spin state.

Resonance Raman spectra of cytochrome P-450cam (P-450cam) and its enzymatically inactive form (P-420) in various oxidation and spin states were measured for the first time. The Raman spectrum of reduced P-450cam was unusual in the sense that the "oxidation-state marker" appeared at an unexpectedly lower frequency (1346 cm-1) in comparison with those of other reduced hemoproteins (approximately 1355-approximately 1365 cm-1), whereas that of oxidized P-450cam was located at a normal frequency. This anomaly in the Raman spectrum of reduced P-450cam can be explained by assuming electron delocalization from the fifth ligand, presumably a thiolate anion, to the antibonding pi orbital of the porphyrin ring. The corresponding Raman line of reduced P-420 appeared at a normal frequency (1360 cm-1), suggesting a status change or replacement of the fifth ligand upon conversion from P-450cam to P-420. The Raman spectrum of reduced P-450cam-metyrapone complex was very similar to that of ferrous cytochrome b5.     

Hydration properties of N-(alpha-hydroxyacyl)-sphingosine: X-ray powder diffraction and FT-Raman spectroscopic studies

The thermotropic properties of N-(alpha-hydroxyacyl)-sphingosine (CER[AS]) in dry and hydrated state were studied by means of X-ray powder diffraction and FT-Raman spectroscopy. The polymorphic states of the CER[AS]/water mixture (lamellar crystalline, lamellar hexagonal gel, liquid crystalline) depend on the thermal pre-treatment of the sample. Only by heating the CER[AS]/water mixture above the melting chain transition can the system be hydrated. At room temperature, both dry and hydrated states form lamellar structures, which differ in their repeat distance and packing of hydrocarbon chains. Above the melting chain transition, hydrated CER[AS] forms a liquid crystalline hexagonal phase, whereas anhydrous CER[AS] forms an isotropic liquid phase. The various phases of hydrated CER[AS] are distinguished on the basis of the corresponding Raman spectra.     

FT-IR,FT-Raman spectra and DFT vibrational analysis of 2-aminobiphenyl

In this work, the Fourier transform infrared (FT-IR) and Fourier transform Raman (FT-Raman) spectra of 2-aminobiphenyl (2ABP) were recorded in the solid phase. The optimised geometry, frequency and intensity of the vibrational bands of 2ABP were obtained by the density functional theory (BLYP and B3LYP) methods with complete relaxation in the potential energy surface using 6-31G(d) basis set. The harmonic vibrational frequencies were calculated and the scaled values have been compared with experimental FT-IR and FT-Raman spectra. The observed and the calculated frequencies are found to be in good agreement. The experimental spectra also coincide satisfactorily with those of theoretically constructed spectrograms.     

Flavin and heme structures in lactate:cytochrome c oxidoreductase: a resonance Raman study

Resonance Raman spectra of Hansenula anomala L-lactate:cytochrome c oxidoreductase (or flavocytochrome b2), of its cytochrome b2 core, and of a bis(imidazole) iron-protoporphyrin complex were obtained at the Soret preresonance from the oxidized and reduced forms. Raman contributions from both the isoalloxazine ring of flavin mononucleotide (FMN) and the heme b2 were observed in the spectra of oxidized flavocytochrome b2. Raman diagrams showing frequency differences of selected FMN modes between aqueous and proteic environments were drawn for various flavoproteins. These diagrams were closely similar for flavocytochrome b2 and for flavodoxins. This showed that the FMN structure must be very similar in both types of proteins, despite their very different proteic pockets. However, the electron density at this macrocycle was found to be higher in flavocytochrome b2 than in these electron transferases. No significant difference was observed between the heme structures in flavocytochrome b2 and in cytochrome b2 core. The porphyrin center-N(pyrrole) distances in the oxidized and reduced heme b2 were estimated to be 1.990 and 2.022 A from frequencies of porphyrin skeletal modes, respectively. The frequency of the vinyl stretching mode of protoporphyrin was found to be very affected in resonance Raman spectra of flavocytochrome b2 and of cytochrome b2 core (1634-1636 cm-1) relative to those observed in the spectra of iron-protoporphyrin [bis(imidazole)] complexes (1620 cm-1). These specificities were interpreted as reflecting a near coplanarity of the vinyl groups of heme b2 with the pyrrole rings to which they are attached. The low-frequency regions of resonance Raman indicated that the iron atoms of the four hemes b2 are in the porphyrin plane whatever their oxidation state. The histidine-Fe-histidine symmetric stretching mode was located at 205 cm-1 in the spectra of flavocytochrome b2 and of cytochrome b2 core. It was insensitive to the iron oxidation state and indicated strong Fe-His bonds in both states.     

Resonance Raman investigations of Escherichia coli-expressed Pseudomonas putida cytochrome P450 and P420.

High-resolution resonance Raman spectra of the ferric, ferrous, and carbonmonoxy (CO)-bound forms of wild-type Escherichia coli-expressed Pseudomonas putida cytochrome P450cam and its P420 form are reported. The ferric and ferrous species of P450 and P420 have been studied in both the presence and absence of excess camphor substrate. In ferric, camphor-bound, P450 (mos), the E. coli-expressed P450 is found to be spectroscopically indistinguishable from the native material. Although substrate binding to P450 is known to displace water molecules from the heme pocket, altering the coordination and spin state of the heme iron, the presence of camphor substrate in P420 samples is found to have essentially no effect on the Raman spectra of the heme in either the oxidized or reduced state. A detailed study of the Raman and absorption spectra of P450 and P420 reveals that the P420 heme is in equilibrium between a high-spin, five-coordinate (HS,5C) form and low-spin six-coordinate (LS,6C) form in both the ferric and ferrous oxidation states. In the ferric P420 state, H2O evidently remains as a heme ligand, while alterations of the protein tertiary structure lead to a significant reduction in affinity for Cys(357) thiolate binding to the heme iron. Ferrous P420 also consists of an equilibrium between HS,5C and LS,6C states, with the spectroscopic evidence indicating that H2O and histidine are the most likely axial ligands. The spectral characteristics of the CO complex of P420 are found to be almost identical to those of a low pH of Mb. Moreover, we find that the 10-ns transient Raman spectrum of the photolyzed P420 CO complex possesses a band at 220 cm-1, which is strong evidence in favor of histidine ligation in the CO-bound state. The equilibrium structure of ferrous P420 does not show this band, indicating that Fe-His bond formation is favored when the iron becomes more acidic upon CO binding. Raman spectra of stationary samples of the CO complex of P450 reveal VFe-CO peaks corresponding to both substrate-bound and substrate-free species and demonstrate that substrate dissociation is coupled to CO photolysis. Analysis of the relative band intensities as a function of photolysis indicates that the CO photolysis and rebinding rates are faster than camphor rebinding and that CO binds to the heme faster when camphor is not in the distal pocket.     

Resonance Raman scattering from hemoproteins. Effects of ligands upon the Raman spectra of various C-type cytochromes.

Resonance Raman spectra were measured for various C-type cytochromes (mammalian cytochrome c, bacterial cytochrome c3, algal photosynthetic cytochrome f, and alkylated cytochrome c) and a B-type cytochrome (cytochrome b5) in their reduced and oxidized states. (1) For ferrous alkylated cytochrome c, a Raman line sensitive to the replacement of an axial ligand of the heme iron uas found around 1540 cm=1. This ligand-sensitive Raman line indicated the transition from acidic (1545 cm-1) to alkaline (1533 cm-1) forms with pK 7.9. The pH dependence of the Raman spectrum corresponded well to that of the optical absorption spectra. (2) For ferrous cytochrome f, the ligand-sensitive Raman line was found at the same frequency as cytochrome c (1545 cm-1). Accordingly two axial ligands are likely to be histidine and methionine as in cytochrome c. (3) For ferrous cytochrome c3, the frequency of the ligand-sensitive Raman line was between those of cytochrome c and cytochrome b5. Since two axial ligands of the heme iron in cytochrome c3 might be histidines. However, a combination of histidine and methionine as a possible set of two axial ligands was not completely excluded for one or two of the four hemes. (4) In ferrous cytochrome b5, two weak Raman lines appeared at 1302 and 1338 cm-1 instead of the strongest band at 1313 cm-1 of C-type ferrous cytochromes. This suggests the practical use of these bands for the identification of types of cytochromes. The difference in frequency and intensity between B- and C-types of hemes implies that the low effective symmetry of the heme in ferrous cytochrome c is due to vibrational coupling of ring modes with peripheral substituents rather than geometrical disortion of heme.     

Effects of cholesterol and adrenodoxin binding on the heme moiety of cytochrome P-450scc: a resonance Raman study

The effects of cholesterol and adrenodoxin binding on resonance Raman spectra of cytochrome P-450scc in both oxidized and CO-reduced states were examined. Upon cholesterol binding, oxidized cytochrome P-450scc showed a significant shift of spin equilibrium from low-spin to high-spin state. Addition of adrenodoxin caused a complete conversion of cholesterol-bound oxidized cytochrome P-450scc to a pure high-spin state that was considered to be in the hexacoordinated state judged by the v10 mode at 1620 cm-1 and v3 mode around 1485 cm-1. Cholesterol in substrate binding site may oppose a linear and perpendicular binding of carbon monoxide to the reduced heme iron, leading to the distorted Fe-C-O linkage. This is based on the following observations: (1) an increase of the Fe-CO stretching frequency to 483 from 477 cm-1 upon addition of cholesterol; (2) an enhanced photodissociability of bound carbon monoxide of CO complex of cytochrome P-450scc in the presence of cholesterol. As another aspect of the effect of cholesterol on the CO complex form of cytochrome P-450scc, the enhanced stability of the native form ("P-450" form) was observed. There was no additional effect of reduced adrenodoxin on the Raman spectra of the CO-reduced form of cytochrome P-450scc.     

Distinct cysteine sulfhydryl environments detected by analysis of Raman S-hh markers of Cys-->Ser mutant proteins

Very little is known about the character or functional relevance of hydrogen-bonded cysteine sulfhydryl (S-H) groups in proteins. The Raman S-H band is a unique and sensitive probe of the local S-H environment. Here, we report the use of Raman spectroscopy combined with site-specific mutagenesis to document the existence of five distinguishable hydrogen-bonded states of buried cysteine sulfhydryl groups in a native protein. The 666 residue subunit of the Salmonella typhimurium bacteriophage P22 tailspike contains eight cysteine residues distributed through the elongated structure. The tailspike cysteine residues display an unusual Raman S-H band complex (2500-2600 cm(-1) interval) indicative of diverse S-H hydrogen-bonding interactions in the native trimeric structure. To resolve specific Cys contributions to the complex Raman band we characterized a set of tailspike proteins with each cysteine replaced by a serine. The mutant proteins, once folded, were structurally and functionally indistinguishable from wild-type tailspikes, except for their Raman S-H signatures. Comparison of the Raman spectra of the mutant and wild-type proteins reveals the following hydrogen-bond classes for cysteine sulfhydryl groups. (i) Cys613 forms the strongest S-H...X bond of the tailspike, stronger than any heretofore observed for a protein. (ii) Cys267, Cys287 and Cys458 form robust S-H...X bonds. (iii) Moderate S-H...X bonding occurs for Cys169 and Cys635. (iv) Cys290 and Cys496 form weak hydrogen bonds. (v) It is remarkable that Cys287 contributes two Raman S-H markers, indicating the population of two distinct hydrogen-bonding states. The sum of the S-H Raman signatures of all eight mutants accurately reproduces the composite Raman band of the wild-type tailspike. The diverse cysteine states may be an outcome of the folding and assembly pathway of the tailspike, which though lacking disulfide bonds in the native state, utilizes transient disulfide bonds in the maturation pathway. This Raman study represents the first detailed assessment of local S-H hydrogen bonding in a native protein and provides information not obtainable directly by other structural probes. The method employed here should be applicable to a wide range of cysteine-containing proteins.