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

ROLANDO CARLOS, ROSSI; SERGIO BENJAMÍN, KAUFMAN; RODOLFO MARTIN GONZALEZ LEBRERO; JENS G, NOERBY; PATRICIO J, GARRAHAN

ANALYTICAL BIOCHEMISTRY

ACADEMIC PRESS INC ELSEVIER SCIENCE

Año: 1999 vol. 270 p. 276 - 285

0003-2697

The present paper describes a quenching-and-wash-ing chamber (QWC) to be used with a rapid-mixingapparatus (RMA) for the study of processes in the mil-lisecond time scale. The QWC enables fast, nonde-structive quenching by cooling and dilution of reac-tants in particulate systems that can be trapped on afilter. The reaction mixture (e.g., at 25°C) is injectedfrom the RMA into the QWC where it is immediatelymixed with a stream of ice-cold solution flowing at arate of 15? 40 ml s ؊1. Quenching requires that the pro-cess studied is slowed considerably by cooling to 0 ?2°Cand/or by removal of reactants by dilution. The equip-ment was characterized through a study of the tightbinding (occlusion) of 86Rb ؉ to purified, membrane-bound Na ؉/K ؉-ATPase. Millipore filters of 0.22? 0.80 ␮mpore size trapped close to 100% of the enzyme protein.Enzyme with occluded 86Rb ؉ was formed in the RMAunder conditions where the rate constant for releaseof Rb ؉ at 25°C is up to 25 s ؊1 and then injected into theQWC. The high off-rate constant is due to the presenceof 2.5 mM ATP, which accelerates release of Rb ؉. Therecovery of occluded 86Rb ؉ on the filter was at least90%, indicating that both cooling of the reactants anddilution of ATP are fast enough to stop the reaction.The quenching time was 3? 4 ms.