An attachment for non-destructive, fast quenching of samples in rapid mixing experiments

R. C. ROSSI; S. B. KAUFMAN; R. M. GONZÁLEZ-LEBRERO; J. G. NØRBY; P. J. GARRAHAN

ANALYTICAL BIOCHEMISTRY

Academic Press

Año: 1999 vol. 270 p. 276 - 285

0003-2697

The present paper describes a quenching-and-washing chamber (QWC) to be used with a rapid-mixing apparatus (RMA) for the study of processes in the millisecond time scale. The QWC enables fast, nondestructive quenching by cooling and dilution of reactantsin particulate systems that can be trapped on a filter. The reaction mixture (e.g., at 25°C) is injected from the RMA into the QWC where it is immediately mixed with a stream of ice-cold solution flowing at a rate of 15–40 ml s21. Quenching requires that the process studied is slowed considerably by cooling to 0–2°C and/or by removal of reactants by dilution. The equipmentwas characterized through a study of the tight binding (occlusion) of 86Rb+ to purified, membrane-bound Na+/K+-ATPase. Millipore filters of 0.22–0.80 mm pore size trapped close to 100% of the enzyme protein. Enzyme with occluded 86Rb1 was formed in the RMAunder conditions where the rate constant for release of Rb+ at 25°C is up to 25 s-1 and then injected into the QWC. The high off-rate constant is due to the presence of 2.5 mM ATP, which accelerates release of Rb+. The recovery of occluded 86Rb+ on the filter was at least 90%, indicating that both cooling of the reactants and dilution of ATP are fast enough to stop the reaction. The quenching time was 3–4 ms.