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

Conducting enzymatic stopped-flow experiments at temperatures far removed from ambient can be very problematic because extremes in temperature (<10 degrees C or >30 degrees 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 degrees 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 degrees C and up to 42 degrees C, and as expected the second-order rate constant (K(1)k(+2)) and the maximum rate of dissociation (k(+2)) both increased with temperature, whereas 1/K-1 is unaffected by the change in temperature. (C) 2015 Elsevier Inc. All rights reserved.