Practical Rapid Quenching Instrument for the Studv of Reaction Mechanisms by Electron Paramagnetic Resonance Spectroscopy I
D. P. Ballou and G. A. Palmer' Department of Biological Chemistry and Biophysics, Research Division, The University of Michigan, A n n Arbor, M i c h 481 0 4
A rapid freezing instrument suitable for the study of fast chemical reactions in aqueous solvents by low temperature Electron Paramagnetic Resonance (EPR) spectroscopy is described. An analysis of its performance reveals that it has a useful time range of 5 msec to 5 sec and therefore is complementary to stopped-flow optical studies. Since the reaction is quenched by freezing, detailed EPR spectral analysis employing long time constants and signal averaging techniques are possible. Using its double mixing capability, the instrument can be used for rapid chemical quenching experiments and for studies of reactions with unstable intermediates. Techniques have also been developed for maintaining anaerobic conditions.
EPR spectroscopy is very useful in characterizing oxidation-reduction reactions since it is often the case that, in certain valence states, one or more of the species involved in the reaction is paramagnetic. When rapid reaction techniques can be utilized with EPR spectroscopy, it is possible to study both spectral and kinetic properties of paramagnetic transient intermediates. However, study by EPR has special requirements. Since many of the transition metals can be observed only a t low temperatures (10-200 OK), the usual flow techniques cannot be generally applied. Furthermore, the insensitivity of EPR measurements (compared with optical methods) requires high concentrations of reactants, many of which are extremely precious. Therefore, in order to be economical in sample consumption and to optimize the signal-to-noise ratio by using slow, and possibly repetitive, scanning techniques, it is necessary to employ a means of stopping the reaction before observation. To meet these requirements, Bray ( I , 2 ) devised the rapid freezing technique which is described in detail elsewhere (3-5). In brief, his apparatus is a hydraulically driven Roughton-Millikan type continuous flow system (6, 7). A length of flexible tubing (the reaction tube) couples the mixing chamber to a nozzle which sprays the reaction mixture into a cryogenic liquid (isopentane a t -140 "C) Present address, D e p a r t m e n t of B i o c h e m i s t r y , R i c e U n i v e r s i ty. Houston, T e x a s 77001.
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(6) (7)
R C Bray Riochem J 81, 189 (1961) R C Bray and R Pettersson Biochem J 81, 194 (1961) R C Bray in Rapid Mixing and Sampling Techniques in Biochemistry B Chance R H Eisenhardt Q H Glbson and K K Lonberg-Holm Ed Academic Press New York N Y 1964 p 195 G Palmer R C Bray and H Beinert J Bioi Chem 239, 2657 (1964) G Palmer and H Beinert in Rapid Mixing and Sampling Techniques in Biochemistry B Chance R H Eisenhardt Q H Gibson and K K Lonberg-Holm Ed Academic Press New York N Y 1964 p 205 F J W Roughton and G A Millikan Proc Roy SOC ( L o n d o n ) A155, 258 (1936) G A Millikan Proc Roy SOC ( L o n d o n ) A155. 277 (19361
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contained in a special EPR sampling tube, thus forming a quenched frozen suspension. The suspension is then packed a t low temperatures into the measuring portion of the EPR tube for subsequent detailed measurements. The age of the sample is determined by the flow rate, the volume of the reaction tube, and the time necessary for quenching. This paper elaborates on improved instrumental aspects of this technique and constitutes the first thorough evaluation of the rapid freezing method. Since the description of the Bray device, several modifications have been designed for rapid quenching. For example, an improved mechanism for the flow system was devised by Hansen and Beinert (8) and mixing improvements as well as an estimation of the quenching time were made by Palmer, Bray, and Beinert ( 4 , 5 ) . A more sophisticated mechanism has since been constructed by Hansen and Beinert which allows a delayed second driving pulse, thus enabling longer reaction times with a minimum consumption of solution (9). A commercial instrument, available from the Durrum Instrument Corp., which can employ multiple mixing as well as a delayed second driving pulse, is useful for chemical quenching and optical observation, but it does not appear suitable for rapid freezing since the required low flow velocities cannot be accurately controlled. A device designed by Lymn et al. ( I O ) for chemical quenching may Be suitable for rapid freezing given certain modifications, while another designed by WBlinder et al. (11) is probably not useful for rapid freezing since it has the same problems as the commercial model. The apparatus described below incorporates all of the performance features just mentioned, is ideal for chemical quenching experiments, and has also made significant improvements in the maintenance of anaerobic conditions. Moreover, the device is simply and economically constructed (Several of these units have been constructed in the University of Michigan Physics Instrument Shop for less than $2000). Detailed drawings useful for building the apparatus are contained in reference (12). Design Criteria. Since relatively high concentrations of samples are required for EPR work, it is desired that most, if not all, of the sample be useful for analysis. A constant flow velocity must be attained in as short a time as possible, maintained for the period of flow required to collect sufficient sample, and terminated in a veg7 short time relative to that of flow. This step-function velocity profile is required to ensure homogeneity of the reaction mixture. A slow acceleration and/or deceleration would contribute sample of unknown age and would constitute (5) R . E. Hansen and H . Beinert, Ana/. Chem , 38, 484 (1986) (9) R . E Hansen and H. Beinert, Madison, Wis. personal communication, 1970. (10) R . W . Lymn G. H Gibson, and J. Hanacek. Rev. Sci. i n s f r u m 4 2 , 356-8 (19711. (11) 0 Walinder. 0. Zetterqvist. and L . Engstrom J Bioi. Chem 244, 1060-64 (1969) (12) D. P. Ballou Thesis (1971). The University of Michigan. University Microfilms, Ann Arbor Mich.. NO.72-14796.
A N A L Y T I C A L C H E M I S T R Y , VOL. 46, NO. 9. AUGUST 1974
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