Temperature manifold for a stopped-flow machine to allow measurements from −10 to +40°C.

Walklate, Jonathan and Geeves, Michael A. (2015) Temperature manifold for a stopped-flow machine to allow measurements from −10 to +40°C. Analytical Biochemistry, 476 . pp. 11-16. ISSN 0003-2697. (doi:https://doi.org/10.1016/j.ab.2015.01.020) (The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided)

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Official URL
http://doi.org/10.1016/j.ab.2015.01.020

Abstract

Conducting enzymatic stopped-flow experiments at temperatures far removed from ambient can be very problematic because extremes in temperature (<10 °C or >30 °C) can damage the machine or the enzyme. We have devised a simple manifold that can be attached to most commercial stopped-flow systems that is independently heated or cooled separate from the main stopped-flow system. Careful calibration of the flow circuit allows the sample to be heated or cooled to the measurement temperature (−8 to +40 °C) 1 to 2 s before mixing in the reaction chamber. This approach allows measurements at temperatures where the stopped flow or the protein is normally unstable. To validate the manifold, we investigated the well-defined ATP-induced dissociation of rabbit muscle myosin subfragment 1 (S1) from its complex with pyrene-labeled actin. This process has both temperature-dependent and -independent components. Use of ethylene glycol allowed us to measure the reaction below 0 °C and up to 42 °C, and as expected the second-order rate constant (K1k+2) and the maximum rate of dissociation (k+2) both increased with temperature, whereas 1/K1 is unaffected by the change in temperature.

Item Type: Article
Uncontrolled keywords: Transient kinetics, fast reactions, physiological temperatures, subzero temperatuers
Subjects: Q Science
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: Sue Davies
Date Deposited: 12 Oct 2015 15:04 UTC
Last Modified: 13 Oct 2015 07:50 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/50887 (The current URI for this page, for reference purposes)
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