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Freeze-Quench Resonance Raman and Electron Paramagnetic Resonance Spectroscopy for Studying Enzyme Kinetics: Application to Azide Binding to Myoglobin

Volume 54, Number 10 (Oct. 2000) Page 1480-1484

Oellerich, Silke; Bill, Eckhard; Hildebrandt, Peter


In the present work we have developed a reliable approach for probing the reaction dynamics of metalloproteins on the millisecond time scale. It is based on the combination of the freeze-quench method with resonance Raman (RR) and electron paramagnetic resonance (EPR) spectroscopy. The reactions are initiated in a mixing chamber and rapidly quenched at low temperature in liquid isopentane after variable delay times. The experimental device is designed in such a way that the same frozen samples can be subsequently studied by two analytical techniques, thereby providing complementary information about the active site structures of intermediate states of the enzyme. In particular, the present setup permits the measurement of high-quality RR spectra despite the interference by the Raman bands of isopentane. With the use of the azide binding reaction to myoglobin as a test case, it is found that RR spectroscopy allows a reliable determination of rate constants, whereas the quantitative analysis of the EPR spectra is associated with a relatively high and unavoidable uncertainty mainly due to irreproducible packing of the freeze-quenched samples.