DEVELOPMENT OF BIOPHYSICAL METHODS FOR STUDY OF BIO-MACROMOLECULAR REACTIONS

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A new inertial drive flow system has been developed for doing quench flow reaction kinetics in the investigation of the mechanism of reaction of Sacroplasmic Reticulum, ATPase, and other enzyme systems which cannot be followed by optical means. In order to use this instrument for Sacroplasmic reticulum investigations, it is necessary to work in what is called the push-push mode. Thus one does a push of the syringes, waits 50 to 500 milliseconds, and pushes again. To achieve this a cam driven lever was developed to allow the hitch feed to advance automatically after each firing. The limit at present is 100 milliseconds, but the system works well at 2 mm i.d. tubing diameter. At 0.5 mm i.d. diameter a materials failure was experienced in the drive mechanism and this is presently being repaired and greatly strengthened. The new hitch feed using a tongue and groove lever action has proven very effective when moved from position to position by hand. Because it provides a positive step as well as drive it has greatly improved the operation of the flow system. A series of test reactions have been run on the thermo stopped-flow giving a dead time of 1.5 msec, a thermistor time resolution of 7.5 msec. and a sensitivity of 100 microdegrees C. Pressure, velocity, and thermal measurements underway over a series of viscosities have provided information needed for the support of the ball mixer theory, i.e. that mixing is mainly the result of changing linear momentum to angular momentum. The results to date demonstrate that for a viscosity of one centipoise a pressure differential of less than 4 psi is sufficient to produce better than 99% mixing at 3 M/sec with thermal fluctuations of only .2 millidegrees C.