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At least 307 records · Page 17Linked to original sources
PubMed2001

ATR-FTIR spectroscopic investigations on the effect of solvents on the permeation of benzoic acid and salicylic acid through silicone membranes.

The effect of a series of alcohols on the permeation of salicylic acid (SA) and benzoic acid (BA) through silicone membrane was evaluated, using Franz-type diffusion cells. Although permeants were applied at the same thermodynamic activity in all vehicles, the resulting fluxes were found to differ significantly. This was a consequence of the interactions between the vehicles and the membrane. The interactions between the vehicles and the membrane were further investigated using ATR-FTIR spectroscopy. With this technique, it was possible to identify two different diffusion processes when the membrane was pre-treated with buffer, whereas one single diffusion process was observed when the membrane was pre-soaked with the vehicle. The technique was successfully used to deconvolute the relative magnitude of partition and diffusion in the permeation process. It was shown that the permeation of both acids was affected by the effect of the vehicles on the diffusion coefficient and the partition coefficient in the silicone membrane. The solubility of the drug in the impregnated membrane was found to be proportional to the saturated solubility in the vehicle used to treat the membrane. The solubility of BA in the impregnated silicone membrane was twice that of SA.

Antifungal Agents↗
PubMed2001

DNA-PK, ATM and ATR as sensors of DNA damage: variations on a theme?

The DNA damage signalling pathway is a core element of the cellular response to genotoxic insult, and its components play key roles in defending against neoplastic transformation. Recent work has indicated that the human ATM and ATR proteins, and their yeast homologues, are intimately involved in sensing DNA damage, suggesting parallels with the DNA double-strand break repair enzyme DNA-PK.

Animals↗
PubMed2001

ATM and ATR: networking cellular responses to DNA damage.

Maintenance of genome stability depends on the appropriate response to DNA damage. This response is based on complex networks of signaling pathways that activate numerous processes and lead ultimately to damage repair and cellular survival - or apoptosis. The protein kinases ATM and ATR are master controllers of some of these networks, acting either in concert or separately to orchestrate the responses to specific types of DNA damage or stalled replication. Understanding their mode of action is essential to our understanding of how cells cope with genotoxic stress.

Apoptosis↗
PubMed2001

"Soft"-cuticle protein secondary structure as revealed by FT-Raman, ATR FT-IR and CD spectroscopy.

The nature of the interaction of insect cuticular proteins and chitin is unknown even though about half of the cuticular proteins sequenced thus far share a consensus region that has been predicted to be the site of chitin binding. We previously predicted the preponderance of a beta-pleated sheet in the consensus region and proposed its responsibility for the formation of helicoidal cuticle (Iconomidou et al., Insect Biochem. Mol. Biol. 29 (1999) 285). In this study, we examined experimentally the secondary structure of intact and guanidine hydrochloride extracted cuticle and the cuticular protein extract. The studied cuticle came from the larval dorsal abdomen of the lepidopteran Hyalophora cecropia, a classical example of "soft" cuticle. Analysis with FT-Raman, ATR FT-IR and CD spectroscopy indicates that antiparallel beta-pleated sheet is the predominant molecular conformation of "soft-cuticle" proteins both in situ in the cuticle and following extraction. It seems that this conformation dictates the modes of chitin-protein interaction in cuticle, in agreement with earlier proposals (Atkins, J. Biosci. 8 (1985) 375).

Amino Acid Sequence↗
PubMed2003

An ATR- and Cdc7-dependent DNA damage checkpoint that inhibits initiation of DNA replication.

We have analyzed how single-strand DNA gaps affect DNA replication in Xenopus egg extracts. DNA lesions generated by etoposide, a DNA topoisomerase II inhibitor, or by exonuclease treatment activate a DNA damage checkpoint that blocks initiation of plasmid and chromosomal DNA replication. The checkpoint is abrogated by caffeine and requires ATR, but not ATM, protein kinase. The block to DNA synthesis is due to inhibition of Cdc7/Dbf4 protein kinase activity and the subsequent failure of Cdc45 to bind to chromatin. The checkpoint does not require pre-RC assembly but requires loading of the single-strand binding protein, RPA, on chromatin. This is the biochemical demonstration of a DNA damage checkpoint that targets Cdc7/Dbf4 protein kinase.

Animals↗
PubMed2001

In situ ATR-FT-IR kinetic studies of molecular transport and surface binding in thin sol-gel films: reactions of chlorosilane reagents in porous silica materials.

ATR-FT-IR spectroscopy was employed to study the kinetics of transport and binding within thin silica sol-gel films. Studies of transport of several nonbinding probe molecules n-heptane, toluene, and 2-propanol, showed that slow diffusion occurs within the micropores of the sol-gel films which could be modeled as a single-exponential accumulation in agreement with numerical models for diffusion in constricted pores. The rate of transport into the film was found to decrease for molecules that interact strongly with the silica surface, which is consistent with adsorption inhibiting the transport of molecules through the pores. In situ spectroscopic studies of surface reactions with diphenylchlorosilane (DP-SiCl) reveal that DPSiCl reacts quickly with surface water to form diphenylhydroxysilane (DPSiOH), the reactive species detected within the film. Analysis of the time-dependent infrared spectra reveals both transport and surface-binding steps in the reaction kinetics. From the magnitudes of the rate constants and the corresponding pure component spectra, it is determined that the surface-binding component is responsible for accumulation of most of the silane at the silica surface. Ex situ spectroscopic studies confirm that Si-O-Si bond formation occurs at room temperature in these sol-gel films. Studies of chlorosilane reactions at silica surfaces pretreated with triethylamine were conducted to investigate the influence of amines on this chemistry; it was determined that the amine enhances the transport of more reagent molecules to the silica surface while the intrinsic rate of the binding reaction is not significantly changed.

Journal Article↗
PubMed2006

Determination of lycopene and beta-carotene content in tomato fruits and related products: Comparison of FT-Raman, ATR-IR, and NIR spectroscopy.

Tomatoes and various products derived from thermally processed tomatoes are major sources of lycopene, but apart from this micronutrient, other carotenoids such as beta-carotene also are present in the fruit. They occur in tomato fruits and various tomato products in amounts of 2.62-629.00 (lycopene) and 0.23-2.83 mg/100 g (beta-carotene). Standard methods for determining the carotenoid content require the extraction of the analyte as well as other cleanup steps. In this work, FT-Raman, ATR-IR, and NIR spectroscopy are applied in order to establish new, fast, and nondestructive calibration methods for quantification of lycopene and beta-carotene content in tomato fruits and related products. The best prediction quality was achieved using a model based on IR spectroscopy (R2 = 0.98 and 0.97, SECV = 33.20 and 0.16 for lycopene and beta-carotene, respectively). In spite of the fact that Raman spectra of tomato products show characteristic key bands of the investigated carotenoids, this method gives slightly lower reliability (R2 = 0.91 and 0.89, SECV = 74.34 and 0.34 for lycopene and beta-carotene, respectively). NIR spectroscopy, which has been used for quantification purposes in the agricultural sector for several decades, in this study shows the worse prediction quality (R2 = 0.85 and 0.80, SECV = 91.19 and 0.41 for lycopene and beta-carotene, respectively).

Carotenoids↗
PubMed2003

Redox-induced transitions in bovine cytochrome bc1 complex studied by perfusion-induced ATR-FTIR spectroscopy.

Redox transitions in a film of detergent-purified bovine cytochrome bc(1) complex were investigated by perfusion-induced attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The technique provides a flexible method for generating redox-induced IR changes of components of bovine cytochrome bc(1) complex at a high signal:noise ratio. These IR redox difference spectra arise from perturbations of prosthetic groups and surrounding protein. Visible difference spectra were recorded synchronously using a light beam reflected from the exposed prism surface and provided a quantitative means of determining the redox transitions that were occurring. IR and visible redox difference spectra of iron-sulfur protein/cytochrome c(1), heme b(H), and heme b(L) were separated by selective reduction and/or oxidation that extends published data on the homologous bacterial enzyme. Several bands could be tentatively assigned to redox-sensitive modes of hemes and ubiquinone and changes in the surrounding protein by comparison with available data for bacterial bc(1) complex, other related heme proteins, and model compounds. Some tentative assignments of further signals to specific amino acids are made on the basis of known crystal structures.

Animals↗
PubMed2003

ATR-FTIR spectroscopy of the P(M) and F intermediates of bovine and Paracoccus denitrificans cytochrome c oxidase.

The structures of P(M) and F intermediates of bovine and Paracoccus denitrificans cytochrome c oxidase were investigated by perfusion-induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. Transitions from the "fast" oxidized state to the P(M) or F states were initiated by perfusion with buffer containing either CO/oxygen or H(2)O(2). Intermediates were quantitated by simultaneous monitoring of visible absorption changes in the protein film. For both bovine and P. denitrificans oxidase, the major features of the IR difference spectrum of P(M) were similar when produced by CO/oxygen or by H(2)O(2) treatments. These IR difference spectra were distinctly different from the IR difference spectrum of F that formed with extended treatment with H(2)O(2). Some IR bands could be assigned tentatively to perturbations of heme a(3) ring modes and substituents, and these perturbations were greater in P(M) than in F. Other bands could be assigned to surrounding protein changes. Strong perturbation of the environment of a carboxylic acid, most likely E-242 (bovine numbering), occurred in P(M) and relaxed back in F. A second redox-sensitive carboxylic acid was also perturbed in the bovine P(M) intermediate. Further consistent signatures of P(M) in both oxidases that were absent in F were strong negative bands at 1547 and 1313 cm(-1) in bovine oxidase (1542 and 1314 cm(-1) in P. denitrificans) and a positive band at approximately 1519 cm(-1). From comparison with available IR data on model compounds, it is suggested that these reflect changes in the covalent tyrosine-histidine ligand to Cu(B). These findings are discussed in relation to the oxidase catalytic cycle.

Animals↗
PubMed2005

Direct observation of redox-linked histidine protonation changes in the iron-sulfur protein of the cytochrome bc1 complex by ATR-FTIR spectroscopy.

The redox-linked protonation chemistry of the iron-sulfur protein (ISP) of the cytochrome bc(1) complex was studied by analysis of the pH dependencies of redox difference spectra using perfusion/electrochemically induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. The ISP of Rhodobacter capsulatus within the intact cytochrome bc(1) complex was analyzed in a mutant form in which the midpoint potential of cytochrome c(1) was lower than that of the ISP. This was compared to a soluble domain of the ISP from the bovine bc(1) complex. Spectra of in situ bacterial and isolated bovine proteins differ markedly only in part of their amide I regions with the in situ protein having additional pH-dependent component(s). Apart from this, both in situ and isolated proteins exhibited the same pH-dependent IR features in reduced minus oxidized difference spectra. Specifically, at high pH, a strong H/D insensitive negative band appeared at 1447/1450 cm(-)(1), together with a peak at 1310 cm(-)(1), the change of a 1267/1255 cm(-)(1) peak/trough into a simple 1266 cm(-)(1) peak, and a trough at 1107 cm(-)(1). Comparison with spectra of model materials indicates that all four signals arise from an imidazolate to imidazole transition of histidine, hence providing the first direct demonstration that histidine is the redox-linked protonation site of the ISP. The 1450 cm(-)(1) band can be assigned to a ring stretch that is unique to the imidazolate form of histidine. It is relatively sharp, has a high extinction coefficient, and provides a novel marker band for the detection of imidazolate intermediates in enzymatic mechanisms generally.

Amino Acid Substitution↗
PubMed2004

ATR-FTIR spectroscopy and isotope labeling of the PM intermediate of Paracoccus denitrificans cytochrome c oxidase.

The structure of the P(M) intermediate of Paracoccus denitrificans cytochrome c oxidase was investigated by perfusion-induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. Transitions from the oxidized to P(M) state were initiated by perfusion with CO/oxygen buffer, and the extent of conversion was quantitated by simultaneously monitoring visible absorption changes. In prior work, tentative assignments of bands were proposed for heme a(3), a change in the environment of the protonated state of a carboxylic acid, and a covalently linked histidine-tyrosine ligand to Cu(B) that has been found in the catalytic site. In this work, reduced minus oxidized difference spectra at pH 6.5 and 9.0 and P(M) minus oxidized difference spectra at pH 9.0 were compared in unlabeled, universally (15)N-labeled, and tyrosine-ring-d(4)-labeled proteins to improve these assignments. In the reduced minus oxidized difference spectrum, (15)N labeling resulted in large changes in the amide II region and a 9 cm(-1) downshift in a 1105 cm(-1) trough that is attributed to histidine. In contrast, changes induced by tyrosine-ring-d(4) labeling were barely detectable where the isotope-sensitive bands are expected. Both isotope substitutions had large effects on P(M) minus oxidized difference spectra. A prominent trough at 1542 cm(-1) was shifted to 1527 cm(-1) with (15)N labeling, and its magnitude was diminished with the appearance of a 1438 cm(-1) trough with tyrosine-ring-d(4) labeling. Both isotope substitutions also had large effects on a 1314 cm(-1) trough in the same spectra. These shifts indicate that the bands are linked to both a nitrogenous compound and a tyrosine, the most obvious candidate being the covalent histidine-tyrosine ligand of Cu(B). Comparison with model material data suggests that the tyrosine hydroxyl group is protonated when the binuclear center is oxidized but deprotonated in the P(M) intermediate. Positive bands at 1519 and 1570 cm(-1) were replaced with bands at 1504 and 1556 cm(-1), respectively, with tyrosine-ring-d(4) labeling, are characteristic of upsilon(7a)(C-O) and upsilon(C-C) bands of neutral phenolic radicals, and most likely reflect the formation of the neutral radical state of the histidine-tyrosine ligand in P(M).

Amino Acids↗
PubMed2004

ATR-FTIR spectroscopy studies of iron-sulfur protein and cytochrome c1 in the Rhodobacter capsulatus cytochrome bc1 complex.

Redox transitions in the Rhodobacter capsulatus cytochrome bc(1) complex were investigated by perfusion-induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy combined with synchronous visible spectroscopy, in both the wild type and a cytochrome c(1) point mutant, M183K, in which the midpoint potential of heme was lowered from the wild-type value of 320 mV to 60 mV. Overall redox difference spectra of the wild type and M183K mutant were essentially identical, indicating that the mutation did not cause any major structural perturbation. Spectra were compared with data on the bovine bc(1) complex, and tentative assignments of several bands could be made by comparison with available data on model compounds and crystallographic structures. The bacterial spectra showed contributions from ubiquinone that were much larger than in the bovine enzyme, arising from additional bound and adventitious ubiquinone. The M183K mutant enabled selective reduction of the iron-sulfur protein which in turn allowed the IR redox difference spectra of ISP and cytochrome c(1) to be deconvoluted at high signal/noise ratios, and features of these spectra are interpreted in light of structural and mechanistic information.

Cytochromes c1↗
PubMed2006

ATR-FTIR redox difference spectroscopy of Yarrowia lipolytica and bovine complex I.

ATR-FTIR spectroscopy in combination with electrochemistry has been applied to the redox centers of Yarrowia lipolytica complex I. The redox spectra show broad similarities with previously published data on Escherichia coli complex I and with new data here on bovine complex I. The spectra are dominated by amide I/II protein backbone changes. Comparisons with redox IR spectra of small model ferredoxins demonstrate that these amide I/II changes arise primarily from characteristic structural changes local to the iron-sulfur centers, rather than from global structural alterations as has been suggested previously. Bands arising from the substrate ubiquinone were evident, as was a characteristic 1405 cm(-)(1) band of the reduced form of the FMN cofactor. Other signals are likely to arise from perturbations or protonation changes of a carboxylic amino acid, histidine, and possibly several other specific amino acids. Redox difference spectra of center N2, together with substrate ubiquinone, were isolated from those of the other iron-sulfur centers by selective redox potentiometry. Its redox-linked amide I/II changes were typical of those in other 4Fe-4S iron sulfur proteins. Contrary to published data on bacterial complex I, no center N2 redox-linked protonation changes of carboxylic amino acids or tyrosine were evident, and other residues that could provide its redox-linked protonation site are discussed. Features of the substrate ubiquinone associated with the center N2 spectrum were particularly clear, with firm assignments possible for bands from both oxidized and reduced forms. This is the first report of IR properties of ubiquinone in complex I, and the data could be used to estimate a stoichiometry of 0.2-0.4 per complex I.

Animals↗
PubMed1997

Orientation in lipid bilayers of a synthetic peptide representing the C-terminus of the A1 domain of shiga toxin. A polarized ATR-FTIR study.

The interaction of a synthetic peptide representing the C-terminal 27 amino acids of the A1 domain of Shiga toxin (residues 220-246) with acidic phospholipid model membranes was characterized by FTIR spectroscopy. This peptide resembles a signal sequence and may mediate the translocation of the catalytic A1 chain of Shiga toxin to the cytoplasm following its retrograde transport to the lumenal compartment of the endoplasmic reticulum (ER). At pH 7 and 5, the peptide underwent a conformational change from random coil to alpha-helix upon addition of negatively charged phospholipids. Examination of the amide II band in the spectrum of the complex at pH 7 and pH 5 showed that in both cases, the N-H groups in the peptide backbone are largely protected from H/D exchange. Using polarized attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) measurements, the orientation of the alpha-helical portion of the peptide was found to be almost perpendicular with respect to the membrane plane at pH 7. However, at pH 5.0-5.4, the alpha-helix axis was preferentially oriented parallel to the membrane plane. The results suggest that at the neutral pH of the ER lumen, the peptide may insert into the membrane, while at the lower pH levels present in earlier endocytic compartments, the peptide would be less likely to traverse the bilayer. In summary, this putative signal peptide may not be able to cause a significant translocation of the A1 domain of Shiga toxin to the cytosol until it reaches the neutral pH of the ER compartment.

Amides↗
PubMed2006

pH dependence and protein selectivity of poly(ethyleneimine)/poly(acrylic acid) multilayers studied by in situ ATR-FTIR spectroscopy.

The selective interaction between polyelectrolyte multilayers (PEM) consecutively adsorbed from poly(ethyleneimine) (PEI) and poly(acrylic acid) (PAC) and a binary mixture containing concanavalin A (COA) and lysozyme (LYZ) based on electrostatic interaction is reported. The composition and structure of the PEM and the uptake of proteins were analyzed by in situ attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy, and the morphology and thickness were characterized by atomic force microscopy (AFM) and ellipsometry. The PEM dissociation degree and charge state and the protein adsorption were shown to be highly dependent on the outermost layer type and the pH in solution. High protein uptake was obtained under electrostatically attractive conditions. This was used to bind selectively one protein from a binary mixture of LYZ/COA. In detail it could be demonstrated that six-layered PEM-6 at pH = 7.3 showed a preferential sorption of positively charged LYZ, while at PEM-5 and pH = 7.3 negatively charged COA could be selectively bound. No protein sorption from the binary mixture was observed at pH = 4.0 for both PEM, when COA, LYZ, and the outermost PEI layer of PEM-5 were positively charged or the outermost PAC layer of PEM-6 was neutral. Furthermore, from factor analysis of the spectral data the higher selectivity was found for PEM-5 compared to PEM-6. Increasing the ionic strength revealed a drastic decrease in the selectivity of both PEM. Evidence was found that the proteins were predominantly bound at the surface and to a minor extent in the bulk phase of PEM. These results suggest possible working regimes and application fields of PEI/PAC multilayer assemblies related to the preparative separation of binary and multicomponent protein mixtures (biofluids, food) as well as to the design of selective protein-resistant surfaces.

Acrylic Resins↗
PubMed1999

In situ monitoring of an escherichia coli fermentation using a diamond composition ATR probe and mid-infrared spectroscopy

A diamond composition ATR probe was used in situ to obtain IR spectra on replicate Escherichia coli fermentations involving a complex medium. The probe showed excellent stability over a 6-month operating period and was unaffected by either agitation or aeration. The formation of an unknown was observed from IR spectra obtained during the sterilization; subsequent experiments proved this to be a reaction product between yeast extract and the phosphates used as buffer salts. Partial-least-squares-based calibration/prediction models were developed for both glucose and acetate using in-process samples. The resulting models had prediction errors of +/-0.26 and +/-0.75 g/L for glucose and acetic acid, respectively, errors which were statistically equivalent to the estimated experimental errors in the reference measurements. Relative concentration profiles for the unknown formed during sterilization could be generated either by tracking peak height at an independent wavelength or by self-modeling curve resolution of the spectral region overlapping that of glucose. These profiles indicated that this compound was metabolized simultaneously with glucose; upon depletion of the glucose, when the microorganism switched to consumption of acetic acid, utilization continued but at a lower rate. The data presented provide an extensive characterization of the performance characteristics of this in situ analysis and clearly demonstrate its utility not just in the quantitative measurement of multiple known species but in the qualitative evaluation of unknown species.

Journal Article↗
PubMed2004

In situ FTIR-ATR analysis and titration of carboxylic acid-terminated SAMs.

FTIR-ATR measurements permit detailed structural analysis and in situ titration of carboxylate-terminated self-assembled monolayers. Both monomeric and dimeric/oligomeric acid groups are seen, and their acid-base behavior is directly monitored. Monomers that are hydrogen bonded only to surrounding water molecules have a pKa = 4.9, while the pKa for the aggregated molecules is 9.3.

Journal Article↗
PubMed2004

Direct detection of formate ligation in cytochrome c oxidase by ATR-FTIR spectroscopy.

The IR signature of binding of formate to the heme a(3-)Cu(B) binuclear site of bovine cytochrome c oxidase has been obtained by perfusion ATR-FTIR spectroscopy. The data show unequivocally that formate binds in its anionic form despite its binding being electroneutral overall. The bound formate can be distinguished from free ligand by the binding-induced sharpening and downshifting of vibrational bands. Formate ligation also causes shifts of vibrational modes of heme a(3) and its substituents and perturbation of histidine residues. The association of the accompanying protonation change with a carboxylate or tyrosine can be ruled out and may involve a histidine metal ligand or, more likely, a simple displacement into the bulk phase of a hydroxide ligand to heme a(3) or CU(B), a reaction which would account for stoichiometric proton uptake and maintenance of net charge within the binuclear center domain.

Animals↗