title: Temperature manifold for a stopped-flow machine to allow measurements from -10 to +40°C.
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 2s 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.
authors: School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK.
authors: Jonathan
authors: J
authors: Walklate
authors: School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK. Electronic address: m.a.geeves@kent.ac.uk.
authors: Michael A
authors: MA
authors: Geeves
mesh_terms: D000818:Animals; D019855:Ethylene Glycol / Q000737:chemistry; D007700:Kinetics; D015879:Myosin Subfragments / Q000737:chemistry; D011817:Rabbits; D013696:Temperature; D013816:Thermodynamics
chemical_list: D015879:Myosin Subfragments; D019855:Ethylene Glycol
keywords: Fast reactions; Physiological temperatures; Subzero temperatures; Transient kinetics