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1.
Raman spectra of amino acids showed complexity in the C-H stretching region (2800-3100 cm(-)(1)) attributed to diversity of CH, CH(2), and CH(3) groups in the side chains, ionization state, and microenvironment. The involvement of specific amino acids in the C-H stretching region of selected proteins, namely, lysozyme, alpha-lactalbumin, beta-lactoglobulin, and their binary mixtures, was investigated by deconvolution using maximum likelihood techniques. The main protein band near 2940 cm(-)(1) was attributed not only to aromatic and aliphatic amino acids but also to many other amino acids. A band near 3065 cm(-)(1) was assigned to aromatic residues, whereas bands near 2880 and 2900 cm(-)(1) corresponded primarily to aliphatic amino acids. Heating at 90 degrees C increased relative intensity near 2940 cm(-)(1) and decreased relative intensity at 2895-2902 cm(-)(1) for lysozyme and its mixtures with alpha-lactalbumin or beta-lactoglobulin. Additional bands at 2812 or 2838 and 3003 cm(-)(1) were observed after heating or in 8 M deuterated urea, reflecting changes upon denaturation.  相似文献   

2.
The interface of 10 or 25% (w/v) bovine serum albumin (BSA), pH 7, buffered solution against mineral or corn oil was studied with a Raman microscope. A gradient of distribution of protein and oil at the interface was observed. The difference spectrum obtained by subtracting the spectrum of mineral or corn oil from that of the BSA/oil interface indicated interactions involving different functional groups of the BSA and the oil molecules. Against mineral oil, the BSA spectrum showed reduced intensity of the tryptophan band at 750 cm(-1) and reduced intensity ratio of the tyrosine doublet at 850-830 cm(-1), indicating changes in the microenvironment of these hydrophobic residues. A negative band at 2850 cm(-1) indicated the involvement of the CH groups in the mineral oil. However, the amide regions, normally assigned to protein secondary structure, were not significantly changed. When the spectrum of BSA was subtracted from the BSA/mineral oil interface spectrum, the resultant difference spectrum showed changes of symmetric and antisymmetric CCC stretches at 980 and 1071 cm(-1), respectively. In contrast, the difference spectrum of BSA/corn oil interface - BSA showed a decrease of CH(2) symmetric stretching at 2850 cm(-1) and a decrease of unsaturated fatty acid hydrocarbon chain stretch at 3010 cm(-1). Raman spectroscopy is a useful tool to study the nature of protein-lipid interactions.  相似文献   

3.
Structural changes of myofibrillar proteins from raw pork muscle and Cantonese sausage at different processing periods were elucidated using Raman spectroscopy. Fourier deconvolution combined with iterative curve fitting were used to analyze the amide I Raman band. Results from amide I, amide III, and C-C stretching vibrations in 890-1060 cm(-1) showed that α-helix decreased accompanied by an increase in β-sheet structure during the first 18 h, and a rebuilding process of secondary structures was observed at the rest stage due to proteolysis. The hierarchical cluster analysis results of amide I and amide III confirmed this rebuilding process. Changes in a doublet near 850 and 830 cm(-1) suggested that some tyrosine residues became buried in a more hydrophobic environment due to intermolecular interactions. Raman spectra in the 2855-2940 cm(-1) region suggested that the environment of aliphatic side chains might have been changed during the final stage and further confirmed above rebuilding process.  相似文献   

4.
Raman spectroscopy was used to elucidate structural changes of beta-lactoglobulin (BLG), whey protein isolate (WPI), and bovine serum albumin (BSA), at 15% concentration, as a function of pH (5.0, 7.0, and 9.0), heating (80 degrees C, 30 min), and presence of 0.24% kappa-carrageenan. Three data-processing techniques were used to assist in identifying significant changes in Raman spectral data. Analysis of variance showed that of 12 characteristics examined in the Raman spectra, only a few were significantly affected by pH, heating, kappa-carrageenan, and their interactions. These included amide I (1658 cm(-1)) for WPI and BLG, alpha-helix for BLG and BSA, beta-sheet for BSA, CH stretching (2880 cm(-1)) for BLG and BSA, and CH stretching (2930 cm(-1)) for BSA. Principal component analysis reduced dimensionality of the characteristics. Heating and its interaction with kappa-carrageenan were identified as the most influential in overall structure of the whey proteins, using principal component similarity analysis.  相似文献   

5.
Lysozyme (25% in D2O, corn oil, and their emulsions (10% w/w oil/D2O solution) were examined by Fourier transform Raman spectroscopy. Emulsions showed three layers, namely, top oil, middle cream, and bottom aqueous layers. Raman spectral analysis revealed hydrophobic interactions involving both protein and lipid components. Compared to lysozyme in D2O, the difference spectrum obtained after subtraction of oil from the cream layer spectrum showed reduced intensity of tryptophan bands at 760, 1013, 1340, and 1360 cm(-)(1), reduced intensity ratio of the tyrosine doublet at 850 and 830 cm(-)(1), and increased intensity of the C-H bending band at 1455 cm(-)(1). Compared to corn oil, the difference spectrum after subtraction of lysozyme from the cream layer spectrum indicated decreased intensity at 2855 cm(-)(1) (lipid CH(2) symmetric stretch) and 3011 cm(-)(1) (unsaturated fatty acid hydrocarbon chain =C-H stretch) and a higher intensity ratio of the C-H stretching band at 2900 cm(-)(1) to bands at 2885 and 2933 cm(-)(1). Spectra of the top and bottom layers resembled corn oil and lysozyme, respectively, except for changes in the D2O band. Raman spectroscopy can be used to detect structural changes in proteins, lipids, and D2O due to protein-lipid interactions.  相似文献   

6.
This paper examines changes in the structure and functionality of fish muscle proteins at frozen storage temperatures known to render very different practical storage lives (-10 and -30 degrees C). Apparent viscosity and dimethylamine (DMA) content showed drastic temperature-related differences during storage. Raman spectroscopy revealed the occurrence of some structural changes involving secondary and tertiary protein structures. The changes in secondary structure were quantified, showing an increase of beta-sheet at the expense of alpha-helix structure. The nuC-H stretching band near 2935 cm(-)(1) increased in intensity, indicating denaturation of the muscle proteins through the exposure of aliphatic hydrophobic groups to the solvent. These structural changes were more pronounced at -10 degrees C but occurred at both storage temperatures, whereas changes in apparent viscosity and DMA only occurred in storage at -10 degrees C. The possible utility of these structural changes for quality assessment is discussed.  相似文献   

7.
pH-Induced cold gelation of whey proteins is a two-step process. After protein aggregates have been prepared by heat treatment, gelation is established at ambient temperature by gradually lowering the pH. To demonstrate the importance of electrostatic interactions between aggregates during this latter process, beta-lactoglobulin aggregates with a decreased iso-electric point were prepared via succinylation of primary amino groups. The kinetics of pH-induced gelation was affected significantly, with the pH gelation curves shifting to lower pH after succinylation. With increasing modification, the pH of gelation decreased to about 2.5. In contrast, unmodified aggregates gel around pH 5. Increasing the iso-electric point of beta-lactoglobulin via methylation of carboxylic acid groups resulted in gelation at more alkaline pH values. Comparable results were obtained with whey protein isolate. At low pH disulfide cross-links between modified aggregates were not formed after gelation and the gels displayed both syneresis and spontaneous gel fracture, in this way resembling the morphology of previously characterized thiol-blocked whey protein isolate gels (Alting, et al., J. Agric. Food Chem. 2000, 48, 5001-5007). Our results clearly demonstrate the importance of the net electric charge of the aggregates during pH-induced gelation. In addition, the absence of disulfide bond formation between aggregates during low-pH gelation was demonstrated with the modified aggregates.  相似文献   

8.
Changes in protein secondary structure and conformation of ovalbumin and beta-lactoglobulin (15% protein w/w) were investigated by Fourier transform Raman spectroscopy and self-deconvolution. The amounts of alpha-helix, beta-sheets, random coil, and beta-turns in native beta-lactoglobulin were 15, 54, 6, and 25%, respectively, and those for ovalbumin (41, 34, 13, and 12%) compared well with published values obtained by X-ray crystallography. The proteins were heated at 90 degrees C for 30 min and high-pressure-treated at 600 MPa for 20 min. Heating increased beta-sheet structures in both proteins at the expense of alpha-helix; for beta-lactoglobulin beta-sheet structures increased from 54 to 70% and for ovalbumin, from 34 to 54%. Random coil increased from 6% in the native protein to 30% in high-pressure-treated beta-lactoglobulin. However, for ovalbumin, the contribution from beta-turns doubled in high-pressure-treated samples, with little change in random coil. Further examination of the deconvoluted amide I band in heated samples revealed several component bands. Bands at 1626 and 1682 cm(-1) for ovalbumin and at 1625 and 1680 cm(-1) for beta-lactoglobulin were observed and are associated with aggregated, intermolecular beta-sheet (beta-aggregation), indicative of heat denaturation. The band seen at 1632-1640 cm(-1) corresponded to intramolecular beta-sheet structures, whereas the band at 1625 cm(-1) is associated with exposed beta-sheets (for example, beta-strands with strong hydrogen bonding that are not part of the core of beta-sheets). In high-pressure-treated samples bands were also observed at 1628 and 1680 cm(-1) for ovalbumin and at 1626 and 1684 cm(-1) for beta-lactoglobulin, suggesting involvement of beta-sheet structures in protein aggregation. Raman bands were observed at 1665-1670 cm(-1) for ovalbumin and at 1663-1675 cm(-1) for beta-lactoglobulin due to random coil structures. The bands at 1650-1660 cm(-1) due to alpha-helices were observed in both heated and high-pressure-treated samples. In addition, in heated samples of both ovalbumin and beta-lactoglobulin, peak intensity increased for beta-sheet in the amide III region, 980-990 cm(-1), and decreased for helix structures (900-960 cm(-1)). In contrast, there was no peak at 1240 cm(-1) (amide III beta-sheet structures) in either high-pressure-treated ovalbumin or beta-lactoglobulin, suggesting that high-pressure denaturation at 600 MPa for 20 min is less extensive than heat denaturation at 90 degrees C for 30 min.  相似文献   

9.
This paper presents an approach to simultaneously analyze polyacetylenes, carotenoids, and polysaccharides in carrot (Daucus carota L.) roots by means of Raman spectroscopy. The components were measured in situ in the plant tissue without any preliminary sample preparation. The analysis is based on the intensive and characteristic key bands observed in the Raman spectrum of carrot root. The molecular structures of the main carrot polyacetylenes, falcarinol and falcarindiol, are similar, but their Raman spectra exhibit specific differences demonstrated by the shift of their -C[triple bond]C- mode from 2258 to 2252 cm(-)(1), respectively. Carotenoids can be identified by -C=C- stretching vibrations (about 1520 and 1155 cm(-)(1)) of the conjugated system of their polyene chain, whereas the characteristic Raman band at 478 cm(-)(1) indicates the skeletal vibration mode of starch molecule. The other polysaccharide, pectin, can be identified by the characteristic band at 854 cm(-)(1), which is due to the -C-O-C- skeletal mode of alpha-anomer carbohydrates. The Raman mapping technique applied here has revealed detailed information regarding the relative distribution of polyacetylenes, carotenoids, starch, and pectin in the investigated plant tissues. The distribution of these components varies among various carrot cultivars, and especially a significant difference can be seen between cultivated carrot and the wild relative D. carota ssp. maritimus.  相似文献   

10.
Fourier transform (FT)-Raman spectroscopy was applied to the analysis of genetically modified (GM) plant tissue. Transgenic carrot callus and tobacco plants possessing a novel StSn1 gene coding for a cysteine-rich snakin-1 polypeptide were obtained after Agrobacterium-mediated transformation. The presence of the StSn1 gene and its expression were confirmed by polymerase chain reactions using plant DNA and cDNA as templates for the amplification of the transgenes. Raman measurements were taken from lyophilized GM carrot callus tissue, fresh GM tobacco leaves, and from seeds produced by GM tobacco plants as well as from the nontransformed controls. Cluster analysis applied to the obtained spectra allowed clear separation of the GM samples expressing the StSn1 gene and the nontransformed control to distinct groups. Such discrimination was achieved only when wavenumber ranges around 500 cm (-1) were analyzed. The results indicate that discrimination between the GM and non-GM materials was related to S-S stretching vibrations in snakin-1, as it contained six sulfur bridges. Other introduced genes, neomycine phosphotransferase ( nptII) and Chitinase ( chit36), did not cause any detectable changes by Raman spectroscopy in plant tissue. This is the first report on the use of Raman spectroscopy for a nondestructive analysis of GM plant material expressing the gene coding for a cysteine-rich polypeptide.  相似文献   

11.
Five monoclonal antibodies (OVA-01, -02, -03, -04, -06) produced against irradiated ovalbumin were investigated in relation to the conformational change in the ovalbumin molecule induced by irradiation with Cobalt-60 gamma-rays. Four antibodies (OVA-01, -02, -04, -06) recognized both native and irradiated ovalbumin, but OVA-03 reacted only with irradiated ovalbumin. These antibodies were classified by modified competitive ELISA, and their K(d) values were determined by the Klotz equation. Epitope analyses were also performed on OVA-03 using CNBr-cleaved peptide fragments from ovalbumin, and it was confirmed that OVA-03 bound to the fragment corresponding to residues Val173-Met196 of the ovalbumin molecule that consists of internal beta-sheet strand 3A and helix F1 containing one open turn. These results demonstrate that dramatic conformational changes in proteins can be induced or that some tertiary or secondary structures can be broken down by gamma-ray irradiation, producing new antigenic sites.  相似文献   

12.
The ultrastructural changes and the main Raman spectral features of water (3100-3500 and 50-600 cm(-)(1) ranges) in frozen-stored hake were studied with the aim of connecting these changes with loss of some functional properties such as water holding capacity, and with modifications of muscle texture. The following results were obtained: (a) The changes in the spaces between myofibrils can be related to modifications of shear resistance. (b) The behavior of the strong 160 cm(-)(1) band can be related to conformational transitions of muscle proteins, to changes in the structure of muscle water, and/or to alterations in protein-water interactions. (c) There were intensity changes in the nu(s)(OH) band that may be attributable to transfer of water to larger spatial domains during frozen storage.  相似文献   

13.
Near-infrared Fourier transform Raman (FT-Raman) spectroscopy was employed to study the molecular structure of edible zein films/coatings, which were fabricated directly from zein protein. The secondary structure of zein protein was mainly in alpha-helix and remained unaltered during film formation as evidenced by the vibrational modes of amide I at 1656 cm(-1) and amide III at 1274 cm(-1). Raman results indicated that hydrophobic interaction played an important role in the formation of zein film and disulfide bonding might be responsible for the structural stability of zein protein during film formation. To enhance its antimicrobial property, an antimicrobial zein film was manufactured by incorporating zein protein with benzoic acid whose structure was then characterized by FT-Raman. It showed that physical entrapment or hydrophobic interaction was crucial to the incorporation of benzoic acid with zein protein, and the secondary structure of the antimicrobial film was still maintained in alpha-helical form. In addition, FT-Raman exhibits its preference in directly determining the thickness of zein films/coatings. By correlating the Raman intensity ratio of nu(1003) to nu(84) (I(1003/84)) versus the thickness of zein film, a linear relationship with high coefficient (R(2) = 0.9927) was obtained, which was then used pragmatically to determine the thickness of zein coatings on apple. It showed that the FT-Raman result (thickness = 0.27 +/- 0.01 mm) was consistent with that of classical micrometric measurement (thickness = 0.28 +/- 0.02 mm). Consequently, FT-Raman provides a direct, simple, and reagent-free method to characterize the structure and the thickness of zein films/coatings.  相似文献   

14.
The effects of heat treatment and dextrin addition on the secondary structure of gliadins were investigated by means of attenuated total reflection Fourier transform infrared spectroscopy (ATR-FT/IR). Gliadins and gliadin/dextrin mixtures (before and after thermal treatment) were prepared as a dried protein film on the ATR-FT/IR zinc selenide cell plate and equilibrated at a water activity (a(w)) of 0.06. The results show that gliadins undergo conformational changes upon thermal treatment both in the absence and in the presence of dextrin. In particular, in the thermally treated gliadins, the decrease of the band at around 1651 cm(-)(1) and the increase of the bands at around 1628 and 1690 cm(-)(1) suggest a loss of alpha-helix structure and a higher content of protein aggregates. The same trend was observed in the presence of dextrin. Concerning the interactions between gliadins and dextrin, gliadin/dextrin mixtures show variations in the amide I region compared to native gliadins (e.g., an increase of the band at 1645 cm(-)(1) and the absence of the band at around 1668 cm(-)(1)) that might be due to hydrogen bond formation between gliadins and dextrin. It was also found that the spectrum of gliadin/dextrin mixtures was less affected by the hydration state than that of native gliadins, as observed from the differential spectra obtained by subtraction of the spectrum obtained at a(w) = 0.06 (driest condition tested) from the spectrum of the sample equilibrated at a(w) = 0.84. This could be due to the fact that C=O and N-H groups of gliadins are engaged to form hydrogen bonds with the hydroxyl groups of dextrin, and so they are not perturbed by the presence of water molecules. Finally, water activity effects on the secondary structure of gliadins are also discussed.  相似文献   

15.
We present a brief introduction to FT‐Raman spectroscopy and examples of its use in cereal science for readers not familiar with Raman spectroscopy. We illustrate the use of FT‐Raman spectroscopy as an analytical tool to measure the degree of chemical modification of chemically modified starches and as a technique to determine conformational and structural changes in plant proteins under different environmental conditions. We briefly describe other applications of Raman spectroscopy in cereal science.  相似文献   

16.
Due to possible contribution of both electrostatic and hydrophobic interactions, use of anionic fluorescent probes such as 1-anilinonaphthalene-8-sulfonic acid (ANS) and cis-parinaric acid (CPA) for the measurement of protein surface hydrophobicity (S0) has been controversial. A neutral probe, 6-propionyl-2-(dimethylamino)-naphthalene (PRODAN), may circumvent this problem. To select the best indicator of S0, in this study, the data for nine model proteins in phosphate buffer, pH 7.5, measured using the above-mentioned probes, was compared to their FT-Raman spectra and calculated solvent accessibility values. Log S0 measured using CPA had the highest correlation (r = 0.874) with the intensities of Raman spectral signals at 760 cm(-1) and 2800-3100 cm(-1), which were combined using a mixture design based on the random-centroid optimization. The order of correlation of Raman spectral parameters with S0 values were CPA > PRODAN > ANS. FT-Raman spectroscopy, therefore, identified CPA, followed by PRODAN, as the fluorescent probe of choice for describing surface hydrophobicity. However, the amino acid surface accessibility calculated using the PredictProtein software was not useful in identifying the best fluorescent probe for the measurement of S0.  相似文献   

17.
根据GenBank公布的猪繁殖与呼吸综合症病毒(Porcine reproductive and respiratory syndrome virus , PRRSV)ORF5基因的核苷酸序列设计3对特异性引物，从重组质粒pMD- ORF5中扩增去除ORF5基因中包括全部信号肽在内的N端跨膜区(28 residues)和中部跨膜区(60 residues)。改造后的ORF5基因分别命名为ORF5-1和 ORF5-2。将2个基因片段定向克隆到原核表达载体pET-32a(+),构建重组质粒pET-ORF5-1和pET-ORF5-2,转化大肠杆菌(Escherichia coli)BL21(DE3)感受态细胞，经IPTG诱导表达和SDS-PAGE电泳分析，ORF5-1和ORF5-2基因获得融合表达，表达量分别为12.2%和39%，证明删除双跨膜区能明显提高ORF5基因的表达量。经Western blot分析，融合蛋白具有一定的免疫学活性，表明摘除跨膜区对该蛋白的抗原性影响不大。  相似文献   

18.
Food plants from the Apiaceae family have been found to demonstrate health-promoting properties. Polyacetylenes are bioactive compounds that are considered to contribute substantially to the beneficial properties of Apiaceae plants. This study applied a Raman mapping technique in the investigation of polyacetylene spatial distribution in fresh roots of some Apiaceae species. Fresh root sections were measured directly without any preliminary preparation. For three Apiaceae species, that is, parsnip ( Pastinaca sativa L.), celeriac ( Apium graveolens var. rapaceum L.), and parsley ( Petroselinum crispum ), the presence of polyacetylenes was confirmed due to the detection of strong and well-separated Raman signals of symmetric -C ≡ C-C ≡ C- stretching vibration in the range of 2200-2300 cm(-1). The spectra were used for generation of two-dimensional maps applying the integration and cluster analysis methods. The Raman maps visualized the distribution of total polyacetylenes as well as individual compounds. Heterogeneous and tissue-specific occurrence of polyacetylenes in roots is shown.  相似文献   

19.
Native beta-lactoglobulin binds and increases the activity of Kluyveromyces lactis beta-galactosidase. Construction of a three-dimensional (3D) model of beta-lactoglobulin showed that lysine residues 15, 47, 69, and 138 are the most exposed ones, thus the ones more likely to interact with beta-galactosidase. Molecular docking estimated the interaction energies of amino acid residues with either lactose or succinic anhydride, showing that Lys(138) is the most likely to react with both. Affinity chromatography demonstrated that succinylated beta-lactoglobulin diminished its ability to bind to the enzyme. Furthermore, when activity was measured in the presence of succinylated beta-lactoglobulin, its activating effect was lost. Since succinylation specifically blocks Lys epsilon-amino groups, their loss very likely causes the disappearance of the activating effect. Results show that the activating effect of beta-lactoglobulin on beta-galactosidase activity is due to the interaction between both proteins and that this interaction is very likely to occur through the Lys epsilon-amino groups of beta-lactoglobulin.  相似文献   

20.
Attenuated total reflectance (ATR) and Fourier transform infrared (FTIR) spectroscopy have been applied in the characterization of sticky dough surfaces. The characterization provides insight in the chemical distribution of gluten protein, starch, water, and fat during dough kneading. ATR is especially useful for selective sampling of dough surfaces because the depth of penetration of radiation is quite shallow. For dough, it is calculated to be in the order of 0.5–4 μm in the mid‐infrared, ideal for measurements of stickiness effects, where only the dough surface is of interest. To investigate the cohesive and adhesive properties of the individual dough constituents, dough was peeled from the ATR plate to study the material that adhered to it. The infrared spectra obtained indicate that fat and gluten protein appear to be located at the outer sticky dough surfaces, rather than water and starch. In comparison with gluten, the fatty component showed relatively strong adhesive forces to the ATR plate; a high residual fraction was measured after peeling the dough. Gluten proteins display different cohesion and adhesion properties that are strongly dependent on their hydration state. This indicates that the degree of hydration of gluten proteins contributes to the sticky properties of (overkneaded) dough. When analyzing gluten protein in D2O instead of a dough matrix, more or less similar results were obtained. Significant differences in amide I and amide II intensities were measured for kneaded and stretched gluten protein in comparison to untreated, wet gluten. Besides changes in the vibrational properties of the amide groups, conformational changes in the tertiary protein structure also were observed. It appears that kneading and stretching of dough results in a major decrease in α‐helices content, accompanied by an increase of extended β‐sheet conformations.  相似文献   

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