Rapid freezing techniques

NiFe] hydrogenase of the purple-sulfur bacterium A. vinosum can exist in various redox states (see Fig. 7.5). Until recently the redox transitions have been studied under equilibrium conditions only. I will shortly report here on the first rapid reactions of H2 and CO with [NiFe] hydrogenases using the so-called rapid-mixing rapid-freezing technique. This technique enables the study of the reaction of an enzyme with its substrate and/or an inhibitor on a timescale down to 10 ms (milliseconds). The basic elements of... 

An EPR signal, characteristic for the superoxide radical, was observed by the rapid-freezing technique in the oxidation at pH 10 of xanthine by dioxygen catalysed by xanthine oxidase (EC 1,2.3.2) The enzymatic reduction of dioxygen by aldehyde oxidase (EC 1.2.3.1) produces also the superoxide radical. 

The conversion of 02 into 02 with release of this ion into the medium is now well-established. The most direct proof is the elegant work of Bray et al. (1977) using their rapid-freeze technique coupled with... 

Rapid-mixing rapid-freezing techniques were developed in the 1960s and 1970s. The development was initiated by workers in the field of redox-enzyme biochemistry—at that time in its infancy—to study the role of transition-metal sites in enzyme catalysis by electron paramagnetic resonance (EPR) spectroscopy see Electron Paramagnetic Resonance (EPR) Spectroscopy). Since EPR spectroscopy of transition metals has to be performed, in general, at low temperatures to slow... 

Finally before discussing each metal in detail, it is necessary to mention the question of measurement temperature. Transition metals often have very fast relaxation times and, whilst copper and molybdenum can in most cases be studied at room temperature, some haemoproteins and many iron-sulphur proteins have to be cooled to close to liquid helium temperatures (4.2°K) for reasonable spectra to be obtained. This means that the nearest we can get to the biological steady state may be by rapidly freezing our samples and that kinetic measurements will also have to involve rapid freeze techniques. Apparatus to do this has been developed but resolution is at present limited to about 5 msec. Since we are studying properties of d electrons, ESR information is in many ways complementary to data obtained from UV/visible spectra where electronic excita-... 

This chapter summarizes some recent developments in the purification of ascorbate oxidase, the number of copper atoms per active molecule, and the stoichiometry of the different copper sites with reference to the classification introduced by Malkin and Malmstrom (5). Furthermore, physical properties of the metal centers are discussed in relation to other simple copper proteins that have been characterized in recent years. Finally, kinetic investigations of ascorbate oxidase reduction are presented as studied by anaerobic stopped-fiow and rapid-freeze techniques. 

Fig. 26 (149). EPR signals of O - obtained both enzymically and non-enzymically. Recording at —170 °C, rapid freezing technique (152) a) xanthine—xanthine oxidase reaction. Note the molybdenum signals (overmodulated). The sloping base line is attributed to an iron signal b) non-enzymically induced Og- from H2O2 and NaIC>4, pH 9.9 c) same as (b) but at pH 13.2...

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