Abstract
A thermal stopped-flow method and its application to biochemical studies are described. The instrument, which was recently constructed for this purpose, consists of a flow-through calorimetric cell, a double two-jet type mixer, and a rapid solution delivery device. Special features of the instrument are: a high sensitivity and a rapid response with the use of a small thermopile consisting of 100 constantan/chromel couples per cm (a thermal sensitivity of 5.9mV K-1 cm-1); a good thermal equilibration between the cell and reactant solutions; and an accurate, reproducible control of flow rate of reacting solution. Using this stopped-flow calorimeter it is now possible to resolve the heat change accompanying a pre-steady state of ATP hydrolysis by myosin into three phases under certain conditions (e. g., at 15°C, pH 7.0 or at 0°C, pH 8.0): a rapid heat burst which is complete before the flow stop; a fairly rapid heat absorption; and a very slow heat production. On the basis of kinetics and other biochemical evidence, these heat changes are assigned respectively to those for ATP binding to myosin, splitting of bound ATP into bound ADP+Pi, and release of Pi from myosin.
