Abstract
Bray’s method [R.C. Bray, Biochem. J. 81. 189–193 (1961)] has been widely used for EPR measurements of freeze trapped enzyme intermediates at low temperature. We have developed a rapid freezing apparatus for XANES and EXAFS measurement in fluorescence mode by applying this method. Solution after mixing is ejected from a nozzle into isopentane kept at -140°C. The ejected solution forms fine ice particles, which are then packed into an observation cell connected with an isopentane vessel by extracting isopentane through a filter. Performance of the apparatus was evaluated on reduction of cytochrome c and deoxygenation of hemoglobin. The packing ratio of frozen samples in the cell was found to be 50 ± 10%, but the intensity of the fluorescence x-ray decreased only by 30%. The quality of the x-ray absorption spectra of rapidly frozen samples was comparable to that of the spectra of samples in the liquid state. The quenching time of the apparatus was estimated to be 5.0 ms. These results demonstrate the feasibility of the rapid-freeze EXAFS method for characterization of intermediates in fast reactions.
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© 1987 Springer-Verlag Berlin Heidelberg
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Saigo, S. et al. (1987). A Rapid-Freeze EXAFS Method for the Studies of Intermediates in Fast Enzymatic Reactions. In: Bianconi, A., Congiu Castellano, A. (eds) Biophysics and Synchrotron Radiation. Springer Series in Biophysics, vol 2. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-71490-0_16
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DOI: https://doi.org/10.1007/978-3-642-71490-0_16
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