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Amide I Band (amide + i_band)
Selected AbstractsPolarized Raman microspectroscopy on intact human hairJOURNAL OF BIOPHOTONICS, Issue 5 2008K. R. Ackermann Abstract Polarization-resolved Raman microspectroscopy with near-infrared laser excitation was applied to intact human hair in order to non-invasively investigate the conformation and orientation of the polypeptide chains. By varying the orientation of the hair shaft relative to the polarization directions of the laser/analyzer, a set of four polarized Raman spectra is obtained; this allows to simultaneously determine both the secondary structure of hair proteins and the orientation of the polypeptide strands relative to the axis of the hair shaft. For the amide I band, results from a quantitative analysis of the polarized Raman spectra are compared with theoretically expected values for fibers with uniaxial symmetry. Based on the polarization behavior of the amide I band and further vibrational bands, a partial ordering of ,-helical polypeptide strands parallel to the hair shaft can be concluded. We suggest that this microspectroscopic approach may be used for human hair diagnostics by detecting structural or orientational alterations of keratins. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] A rapid method for assessing lipid:protein and detergent:protein ratios in membrane-protein crystallizationACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2003Corrie J. B. DaCosta A simple procedure for rapidly measuring lipid:protein ratios and detergent concentrations at different stages of the solubilization, purification and crystallization of membrane proteins has been developed. Fourier-transform infrared spectra recorded from 10,µl aliquots of solution using a single-bounce diamond-attenuated total reflectance apparatus exhibit characteristic bands arising from the vibrations of lipid, protein and detergent. Lipid:protein molar ratios as low as 5:1 (for a protein with a molecular weight of 300,kDa) are determined by comparing the ratio of the integrated intensity of the lipid ester carbonyl band near 1740,cm,1 with the protein amide I band near 1650,cm,1. Detergent concentrations at levels well below the critical micellar concentration of most detergents are determined by comparing the integrated intensities of the detergent vibrations, particularly in the 1200,1000,cm,1 region, with a standard curve. Protein amide I band-shape analysis provides insight into the effects of detergents on protein secondary structure. The importance of monitoring detergent concentration changes during simple procedures, such as the concentration of a membrane protein by ultrafiltration, is demonstrated. This analytical tool has been used to rapidly establish protocols for minimizing lipid and detergent levels in solubilized membrane-protein samples. [source] Effects of ELF magnetic field on membrane protein structure of living HeLa cells studied by Fourier transform infrared spectroscopyBIOELECTROMAGNETICS, Issue 7 2003Toshitaka Ikehara Abstract The effects of exposure to a 50 Hz magnetic field (maximum of 41.7 to 43.6 mT) on the membrane protein structures of living HeLa cells were studied using attenuated total reflection infrared spectroscopy. One min of such exposure shifted peak absorbance of the amide I band to a smaller wave number, reduced peak absorbance of the amide II band, and increased absorbance at around 1600 cm,1. These results suggest that exposure to the ELF magnetic field has reversible effects on the N,H inplane bending and C,N stretching vibrations of peptide linkages, and changes the secondary structures of ,-helix and ,-sheet in cell membrane proteins. Bioelectromagnetics 24:457,464, 2003. © 2003 Wiley-Liss, Inc. [source] Spectroscopic study on structure of horseradish peroxidase in water and dimethyl sulfoxide mixtureBIOPOLYMERS, Issue 2 2002Yasushi Maeda Abstract The structure of horseradish peroxidase (HRP) in phosphate buffered saline (PBS)/dimethyl sulfoxide (DMSO) mixed solvents at different compositions is investigated by IR, electronic absorption, and fluorescence spectroscopies. The fluorescence spectra and the amide I spectra of ferric HRP [HRP(Fe3+)] show that overall structural changes are relatively small up to 60% DMSO. Although the amide I band of HRP(Fe3+) shows a gradual change in the secondary structure and a decrease in the contents of , helices, its fluorescence spectra indicate that the distance between the heme and Trp173 is almost constant. In contrast, the changes in the positions of the Soret bands for resting HRP(Fe3+) and catalytic intermediates (compounds I and II) and the IR spectra at the CO stretching vibration mode of carbonyl ferrous HRP [HRP(Fe2+)-CO] show that the microenvironment in the distal heme pocket is altered, even with low DMSO contents. The large reduction of the catalytic activity of HRP even at low DMSO contents can be attributed to the structural transition in the distal heme pocket. In PBS/DMSO mixtures containing more than 70 vol % DMSO, HRP undergoes large structural changes, including a large loss of the secondary structure and a dissociation of the heme from the apoprotein. The presence of the components of the amide I band that can be assigned to strongly hydrogen bonding amide CO groups at 1616 and 1684 cm,1 suggests that the denatured HRP may aggregate through strong hydrogen bonds. © 2002 John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 67: 107,112, 2002 [source] New Fourier transform infrared based computational method for peptide secondary structure determination.BIOPOLYMERS, Issue 2 2001Abstract Fourier transform infrared (FTIR) experiments in dimethylsulfoxide, a solvent incapable of H donation, demonstrate that H , D isotopic replacement on the amide side of peptide bonds involves modifications of both the position and intensity of the amide I band. The effect of the isotopic substitution is particularly significant in the 1710,1670 and 1670,1650 cm,1 regions, which are generally associated with ,-turns and ,-helices. This behavior, attributed to the existence of intramolecular H-bonds in the polypeptide chain, is directly correlated to the presence of different secondary structures. Utilizing the effects induced by isotopic substitution, a method for the quantitative determination of the percentage of intramolecular H-bonds and the correlated secondary structures is proposed. The method consists of three principal steps: resolution of the fine structure of the amide I band with the determination of the number and position of the different components; reconstruction of the experimentally measured amide I band as a combination of Gaussian and Lorentzian functions, centered on the wave numbers set by band-narrowing methods, through a curve-fitting program; and quantitative determination of the population of the H-bonded carbonyls and the correlated secondary structures by comparison of the integrated intensities pertaining to the components with homologous wave numbers before and after isotopic exchange. The method is tested on a synthetic fragment of proocytocin that was previously analyzed by NMR techniques using the same solvent systems. © 2001 John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 62: 95,108, 2001 [source] Rapid monitoring of recombinant antibody production by mammalian cell cultures using fourier transform infrared spectroscopy and chemometricsBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2010Christopher A. Sellick Abstract Fourier transform infrared (FT-IR) spectroscopy combined with multivariate statistical analyses was investigated as a physicochemical tool for monitoring secreted recombinant antibody production in cultures of Chinese hamster ovary (CHO) and murine myeloma non-secreting 0 (NS0) cell lines. Medium samples were taken during culture of CHO and NS0 cells lines, which included both antibody-producing and non-producing cell lines, and analyzed by FT-IR spectroscopy. Principal components analysis (PCA) alone, and combined with discriminant function analysis (PC-DFA), were applied to normalized FT-IR spectroscopy datasets and showed a linear trend with respect to recombinant protein production. Loadings plots of the most significant spectral components showed a decrease in the C,O stretch from polysaccharides and an increase in the amide I band during culture, respectively, indicating a decrease in sugar concentration and an increase in protein concentration in the medium. Partial least squares regression (PLSR) analysis was used to predict antibody titers, and these regression models were able to predict antibody titers accurately with low error when compared to ELISA data. PLSR was also able to predict glucose and lactate amounts in the medium samples accurately. This work demonstrates that FT-IR spectroscopy has great potential as a tool for monitoring cell cultures for recombinant protein production and offers a starting point for the application of spectroscopic techniques for the on-line measurement of antibody production in industrial scale bioreactors. Biotechnol. Bioeng. 2010; 106: 432,442. © 2010 Wiley Periodicals, Inc. [source] | |||||||||||||