title: Microsecond freeze-hyperquenching: development of a new ultrafast micro-mixing and sampling technology and application to enzyme catalysis.
abstract: A novel freeze-quench instrument with a characteristic <<dead-time>> of 137 +/- 18 micros is reported. The prototype has several key features that distinguish it from conventional freeze-quench devices and provide a significant improvement in time resolution: (a) high operating pressures (up to 400 bar) result in a sample flow with high linear rates (up to 200 m s(-1)); (b) tangential micro-mixer with an operating volume of approximately 1 nl yields short mixing times (up to 20 micros); (c) fast transport between the mixer and the cryomedium results in short reaction times: the ageing solution exits the mixer as a free-flowing jet, and the chemical reaction occurs "in-flight" on the way to the cryomedium; (d) a small jet diameter (approximately 20 microm) and a high jet velocity (approximately 200 m s(-1)) provide high sample-cooling rates, resulting in a short cryofixation time (up to 30 micros). The dynamic range of the freeze-quench device is between 130 micros and 15 ms. The novel tangential micro-mixer efficiently mixes viscous aqueous solutions, showing more than 95% mixing at eta < or = 4 (equivalent to protein concentrations up to 250 mg ml(-1)), which makes it an excellent tool for the preparation of pre-steady state samples of concentrated protein solutions for spectroscopic structure analysis. The novel freeze-quench device is characterized using the reaction of binding of azide to metmyoglobin from horse heart. Reaction samples are analyzed using 77 K optical absorbance spectroscopy, and X-band EPR spectroscopy. A simple procedure of spectral analysis is reported that allows (a) to perform a quantitative analysis of the reaction kinetics and (b) to identify and characterize novel reaction intermediates. The reduction of dioxygen by the bo3-type quinol oxidase from Escherichia coli is assayed using the MHQ technique. In these pilot experiments, low-temperature optical absorbance measurements show the rapid oxidation of heme o3 in the first 137 micros of the reaction, accompanied by the formation of an oxo-ferryl species. X-band EPR spectroscopy shows that a short-living radical intermediate is formed during the oxidation of heme o3. The radical decays within approximately 1 ms concomitant with the oxidation of heme b, and can be attributed to the PM reaction intermediate converting to the oxoferryl intermediate F. The general field of application of the freeze-quench methodology is discussed.
authors.affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Cherepanov
authors.forename: Simon
authors.initials: S
authors.lastname: De Vries
mesh_terms: D001386:Azides / Q000737:chemistry / Q000378:metabolism; D001671:Biochemistry / Q000295:instrumentation* / Q000379:methods*; D002384:Catalysis; D004798:Enzymes / Q000032:analysis / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005615:Freezing; D008786:Metmyoglobin / Q000737:chemistry / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods; D013696:Temperature; D013997:Time Factors
chemical_list: D001386:Azides; D004798:Enzymes; D008786:Metmyoglobin; D010088:Oxidoreductases; C020094:duroquinol oxidase; D010100:Oxygen