New insights into conformational and functional stability of human α-thrombin probed by high hydrostatic pressure

摘要: The effects of high hydrostatic pressure (HHP) and urea on conformational transitions human a-thrombin structure were studied by fluorescence spectroscopy measuring the catalytic activity enzyme. Treatment thrombin with produced a progressive red shift in center mass intrinsic emission spectrum, maximum displacement 650 cm )1 . HHP (270 MPa) shifted centre only 370 combined subdenaturing concentration (1.5 M) displaced � 750 binding fluorescent probe bis(8-anilinonaphthalene-1-sulfonate) to was increased 1.8-, 4.0-, 2.7-fold after treatment concentration, or urea, respectively, thus suggesting that all treatments convert enzyme partially folded intermediates exposed hydrophobic regions. On other hand, (but not HHP) dithiothreitol progressively probe, this condition converts completely unfolded state. Urea also led different conformations when changes site environment assessed using fluorescein-D-Phe-ProArg-cloromethylketone-a-thrombin: addition up 2 M gradually decreased 65% initial intensity, whereas caused increase fluorescence. Hydrolysis synthetic substrate S-2238 enhanced (35%) abolished at higher concentrations, while inhibited enzyme’s 45% it 1.5 present. Altogether, analysis indicates formation dissimilar intermediate states during denaturation these agents.