TAILIEUCHUNG - Báo cáo khóa học: New insights into conformational and functional stability of human a-thrombin probed by high hydrostatic pressure

The effects of high hydrostatic pressure (HHP) and urea on conformational transitions of humana-thrombin structure were studiedbyfluorescence spectroscopy andbymeasuring the catalytic activity of the enzyme. Treatment of thrombin with urea produced a progressive red shift in the center of mass of the intrinsic fluorescence emission spectrum, with a maximum displacement of 650 cm )1 . HHP (270 MPa) shifted the centre ofmass by only 370 cm )1 .HHP combined with a subdenaturing urea concentration () displaced the centre of mass by 750 cm )1 | Eur. J. Biochem. 271 3580-3587 2004 FEBS 2004 doi New insights into conformational and functional stability of human a-thrombin probed by high hydrostatic pressure Luis Mauricio T. R. Lima1 Russolina B. Zingali2 Debora Foguel2 and Robson Q. Monteiro2 1 Departamento de Medicamentos Faculdade de Farmacia and 2Departamento de Bioquimica Medica Instituto de Ciências Biomedicas Universidade Federal do Rio de Janeiro Brazil The effects of high hydrostatic pressure HHP and urea on conformational transitions of human a-thrombin structure were studied by fluorescence spectroscopy and by measuring the catalytic activity of the enzyme. Treatment of thrombin with urea produced a progressive red shift in the center of mass of the intrinsic fluorescence emission spectrum with a maximum displacement of 650 cm-1. HHP 270 MPa shifted the centre of mass by only 370 cm-1. HHP combined with a subdenaturing urea concentration m displaced the centre of mass by w 750 cm-1. The binding of the fluorescent probe bis 8-anilinonaphthalene-1-sulfonate to thrombin was increased by and after treatment with high urea concentration HHP or HHP combined with urea respectively thus suggesting that all treatments convert the enzyme to partially folded intermediates with exposed hydrophobic regions. On the other hand treatment of thrombin with urea but not HHP combined with dithiothreitol progressively displaced the fluorescent probe thus suggesting that this condition converts the enzyme to a completely unfolded state. Urea and HHP also led to different conformations when changes in the thrombin catalytic site environment were assessed using the fluorescence emission of fluorescein-D-Phe-Pro-Arg-cloromethylketone-a-thrombin addition of urea up to 2 M gradually decreased the fluorescence emission of the probe to 65 of the initial intensity whereas HHP caused a progressive increase in fluorescence. Hydrolysis of the synthetic substrate S-2238 was .

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