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Henry W. Strobel - One of the best experts on this subject based on the ideXlab platform.
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Photoaffinity Labeling of Cytochrome P4501A1 with AzidoCumene: Identification of Cumene Hydroperoxide Binding Region
Archives of Biochemistry and Biophysics, 2002Co-Authors: Tomas Cvrk, Henry W. StrobelAbstract:Abstract Cumene hydroperoxide can support cytochrome P450-catalyzed reactions in the absence of molecular oxygen, NADPH, and cytochrome P450-NADPH oxidoreductase. Its binding at the cytochrome P450 active site is governed by the structure of the Cumene hydroperoxide binding region. In order to define the region of cytochrome P4501A1 at which Cumene hydroperoxide binds, we prepared an analog of Cumene hydroperoxide for use as a photoaffinity label. p -Azido-isopropylbenzene (azidoCumene) and its tritiated derivative were photolyzed in water solution by uv light with a half-life of 29 s. The 7-ethoxycoumarin deethylatation catalyzed by P450 using the Cumene hydroperoxide-supported system was strongly inhibited by the presence of the label. Covalent binding to the protein after photoactivation was blocked by 50% in the presence of Cumene hydroperoxide. HPLC analysis after trypsin digestion of the labeled protein showed that [ 3 H]azidoCumene was attached covalently to the peptide VDMTPAYGLTLK corresponding to residues 492–503 in the 1A1 sequence. The radioactivity level of this fraction was reduced by 50% when the labeling was carried out in the presence of Cumene hydroperoxide. To confirm the identified region the labeled protein was cleaved by cyanogen bromide. HPLC separation of the CNBr digest showed two peaks with a high level of radioactivity. The SDS/Tricine PAGE analysis of the radioactive fraction with an elution time of 43 min revealed a 2.4-kDa peptide carrying a high level of covalently bound radioactivity. The N-terminal sequence identified the labeled peptide to be a fragment generated by CNBr corresponding to residues 494–512. The N-terminal sequence of the labeled peptide with elution time of 27 min, TLKH, matches amino acid residues 501–504 in the P4501A1 sequence. We can conclude that in the overlapping region of all three identified peptides, T501–L502–K503, is the site where azidoCumene covalently binds to P4501A1. The sequence alignment of cytochrome P4501A1 with cytochrome P450102 predicts that this region might correspond to β-sheet structure localized on the distal side of the heme ring near the I helix and the oxygen binding pocket. To our knowledge, this is the first report to localize the Cumene hydroperoxide binding region in the cytochrome P450 active site.
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Photoaffinity Labeling of Cytochrome P4501A1 with AzidoCumene: Identification of Cumene Hydroperoxide Binding Region
Archives of Biochemistry and Biophysics, 2002Co-Authors: Tomas Cvrk, Henry W. StrobelAbstract:Abstract Cumene hydroperoxide can support cytochrome P450-catalyzed reactions in the absence of molecular oxygen, NADPH, and cytochrome P450-NADPH oxidoreductase. Its binding at the cytochrome P450 active site is governed by the structure of the Cumene hydroperoxide binding region. In order to define the region of cytochrome P4501A1 at which Cumene hydroperoxide binds, we prepared an analog of Cumene hydroperoxide for use as a photoaffinity label. p -Azido-isopropylbenzene (azidoCumene) and its tritiated derivative were photolyzed in water solution by uv light with a half-life of 29 s. The 7-ethoxycoumarin deethylatation catalyzed by P450 using the Cumene hydroperoxide-supported system was strongly inhibited by the presence of the label. Covalent binding to the protein after photoactivation was blocked by 50% in the presence of Cumene hydroperoxide. HPLC analysis after trypsin digestion of the labeled protein showed that [ 3 H]azidoCumene was attached covalently to the peptide VDMTPAYGLTLK corresponding to residues 492–503 in the 1A1 sequence. The radioactivity level of this fraction was reduced by 50% when the labeling was carried out in the presence of Cumene hydroperoxide. To confirm the identified region the labeled protein was cleaved by cyanogen bromide. HPLC separation of the CNBr digest showed two peaks with a high level of radioactivity. The SDS/Tricine PAGE analysis of the radioactive fraction with an elution time of 43 min revealed a 2.4-kDa peptide carrying a high level of covalently bound radioactivity. The N-terminal sequence identified the labeled peptide to be a fragment generated by CNBr corresponding to residues 494–512. The N-terminal sequence of the labeled peptide with elution time of 27 min, TLKH, matches amino acid residues 501–504 in the P4501A1 sequence. We can conclude that in the overlapping region of all three identified peptides, T501–L502–K503, is the site where azidoCumene covalently binds to P4501A1. The sequence alignment of cytochrome P4501A1 with cytochrome P450102 predicts that this region might correspond to β-sheet structure localized on the distal side of the heme ring near the I helix and the oxygen binding pocket. To our knowledge, this is the first report to localize the Cumene hydroperoxide binding region in the cytochrome P450 active site.
Roberto Sanchirico - One of the best experts on this subject based on the ideXlab platform.
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thermal decomposition of Cumene hydroperoxide chemical and kinetic characterization
Aiche Journal, 2008Co-Authors: I Di Somma, Roberto Andreozzi, Marisa Canterino, V Caprio, Roberto SanchiricoAbstract:Thermal decomposition process of Cumene hydroperoxide (CHP) in Cumene has been investigated by different researchers from chemical and kinetic point of view although its reactive characteristics have not yet been fully identified. The major discrepancy in the literature data is represented by different reaction nth-order and autocatalytic kinetics suggested. In the present work CHP thermal decomposition is studied by means of isothermal experiments, adiabatic and scanning calometry on commercial hydroperoxide samples. The most simple autocatalytic scheme is adopted for the analysis of the data collected on CHP solutions in Cumene at different initial concentrations (80%–30% w/w) allowing to estimate the kinetic parameters regulating the process. A characterization of the intermediate and product distribution at varying reaction time is attempted. The effect on the system reactivity of the addition of small quantities of carbinol or acetophenone or a-methylstyrene to the CHP solution in Cumene is also studied. 2008 American Institute of Chemical Engineers AIChE J, 54: 1579–1584, 2008
Michael T. K. Ling - One of the best experts on this subject based on the ideXlab platform.
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Kinetics of catalyst-free thermal and photo-oxidation of Cumene
Journal of Thermal Analysis and Calorimetry, 2013Co-Authors: Vadim V. Krongauz, John F. O’connell, Michael T. K. LingAbstract:Kinetics of thermal and photo-oxidation of Cumene in the absence of catalyst was studied using high-pressure differential scanning calorimetry and low-pressure photocalorimetry. Kinetics of oxidation was followed by Cumene hydroperoxide (CHP), acetophenone, and phenol formation. The amount of CHP formed was deduced from the total heat of reaction of thermal degradation of CHP at 453 K and using a new gas chromatographic method. CHP solution in Cumene oxidized at 453 K and 680 psi of oxygen reproducibly with the heat of reaction linearly dependent on peroxide concentration in Cumene. It was confirmed that Cumene thermal oxidation was slow at
Tomas Cvrk - One of the best experts on this subject based on the ideXlab platform.
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Photoaffinity Labeling of Cytochrome P4501A1 with AzidoCumene: Identification of Cumene Hydroperoxide Binding Region
Archives of Biochemistry and Biophysics, 2002Co-Authors: Tomas Cvrk, Henry W. StrobelAbstract:Abstract Cumene hydroperoxide can support cytochrome P450-catalyzed reactions in the absence of molecular oxygen, NADPH, and cytochrome P450-NADPH oxidoreductase. Its binding at the cytochrome P450 active site is governed by the structure of the Cumene hydroperoxide binding region. In order to define the region of cytochrome P4501A1 at which Cumene hydroperoxide binds, we prepared an analog of Cumene hydroperoxide for use as a photoaffinity label. p -Azido-isopropylbenzene (azidoCumene) and its tritiated derivative were photolyzed in water solution by uv light with a half-life of 29 s. The 7-ethoxycoumarin deethylatation catalyzed by P450 using the Cumene hydroperoxide-supported system was strongly inhibited by the presence of the label. Covalent binding to the protein after photoactivation was blocked by 50% in the presence of Cumene hydroperoxide. HPLC analysis after trypsin digestion of the labeled protein showed that [ 3 H]azidoCumene was attached covalently to the peptide VDMTPAYGLTLK corresponding to residues 492–503 in the 1A1 sequence. The radioactivity level of this fraction was reduced by 50% when the labeling was carried out in the presence of Cumene hydroperoxide. To confirm the identified region the labeled protein was cleaved by cyanogen bromide. HPLC separation of the CNBr digest showed two peaks with a high level of radioactivity. The SDS/Tricine PAGE analysis of the radioactive fraction with an elution time of 43 min revealed a 2.4-kDa peptide carrying a high level of covalently bound radioactivity. The N-terminal sequence identified the labeled peptide to be a fragment generated by CNBr corresponding to residues 494–512. The N-terminal sequence of the labeled peptide with elution time of 27 min, TLKH, matches amino acid residues 501–504 in the P4501A1 sequence. We can conclude that in the overlapping region of all three identified peptides, T501–L502–K503, is the site where azidoCumene covalently binds to P4501A1. The sequence alignment of cytochrome P4501A1 with cytochrome P450102 predicts that this region might correspond to β-sheet structure localized on the distal side of the heme ring near the I helix and the oxygen binding pocket. To our knowledge, this is the first report to localize the Cumene hydroperoxide binding region in the cytochrome P450 active site.
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Photoaffinity Labeling of Cytochrome P4501A1 with AzidoCumene: Identification of Cumene Hydroperoxide Binding Region
Archives of Biochemistry and Biophysics, 2002Co-Authors: Tomas Cvrk, Henry W. StrobelAbstract:Abstract Cumene hydroperoxide can support cytochrome P450-catalyzed reactions in the absence of molecular oxygen, NADPH, and cytochrome P450-NADPH oxidoreductase. Its binding at the cytochrome P450 active site is governed by the structure of the Cumene hydroperoxide binding region. In order to define the region of cytochrome P4501A1 at which Cumene hydroperoxide binds, we prepared an analog of Cumene hydroperoxide for use as a photoaffinity label. p -Azido-isopropylbenzene (azidoCumene) and its tritiated derivative were photolyzed in water solution by uv light with a half-life of 29 s. The 7-ethoxycoumarin deethylatation catalyzed by P450 using the Cumene hydroperoxide-supported system was strongly inhibited by the presence of the label. Covalent binding to the protein after photoactivation was blocked by 50% in the presence of Cumene hydroperoxide. HPLC analysis after trypsin digestion of the labeled protein showed that [ 3 H]azidoCumene was attached covalently to the peptide VDMTPAYGLTLK corresponding to residues 492–503 in the 1A1 sequence. The radioactivity level of this fraction was reduced by 50% when the labeling was carried out in the presence of Cumene hydroperoxide. To confirm the identified region the labeled protein was cleaved by cyanogen bromide. HPLC separation of the CNBr digest showed two peaks with a high level of radioactivity. The SDS/Tricine PAGE analysis of the radioactive fraction with an elution time of 43 min revealed a 2.4-kDa peptide carrying a high level of covalently bound radioactivity. The N-terminal sequence identified the labeled peptide to be a fragment generated by CNBr corresponding to residues 494–512. The N-terminal sequence of the labeled peptide with elution time of 27 min, TLKH, matches amino acid residues 501–504 in the P4501A1 sequence. We can conclude that in the overlapping region of all three identified peptides, T501–L502–K503, is the site where azidoCumene covalently binds to P4501A1. The sequence alignment of cytochrome P4501A1 with cytochrome P450102 predicts that this region might correspond to β-sheet structure localized on the distal side of the heme ring near the I helix and the oxygen binding pocket. To our knowledge, this is the first report to localize the Cumene hydroperoxide binding region in the cytochrome P450 active site.
I Di Somma - One of the best experts on this subject based on the ideXlab platform.
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thermal decomposition of Cumene hydroperoxide chemical and kinetic characterization
Aiche Journal, 2008Co-Authors: I Di Somma, Roberto Andreozzi, Marisa Canterino, V Caprio, Roberto SanchiricoAbstract:Thermal decomposition process of Cumene hydroperoxide (CHP) in Cumene has been investigated by different researchers from chemical and kinetic point of view although its reactive characteristics have not yet been fully identified. The major discrepancy in the literature data is represented by different reaction nth-order and autocatalytic kinetics suggested. In the present work CHP thermal decomposition is studied by means of isothermal experiments, adiabatic and scanning calometry on commercial hydroperoxide samples. The most simple autocatalytic scheme is adopted for the analysis of the data collected on CHP solutions in Cumene at different initial concentrations (80%–30% w/w) allowing to estimate the kinetic parameters regulating the process. A characterization of the intermediate and product distribution at varying reaction time is attempted. The effect on the system reactivity of the addition of small quantities of carbinol or acetophenone or a-methylstyrene to the CHP solution in Cumene is also studied. 2008 American Institute of Chemical Engineers AIChE J, 54: 1579–1584, 2008
