This proposal relates to the emerging technique of high precision ultrasonic velocimetry that has provided unique and remarkable insights on biomolecular interactions.Often much of the driving force for macromolecular associations comes from reorganization of bound water. The unique sensitivity of sound velocity to changes in hydration makes it a powerful technique which can complement and sometimes surpass techniques such as isothermal titration calorimetry. Sound velocity monitored titrations can measure the affinity of binding of a macromolecule to a wide range of compounds, including drugs and other macromolecules. Exploitation of the technique has been hindered by the absence of commercial instrumentation with the precision, accuracy, sensitivity, and small sample volume required for biochemical studies. The goal of this proposal is to develop an ultrasonic velocimeter to analyze molecular association processes of biochemical and/or pharmaceutical importance. A prototype instrument using a recently patented acoustic resonator, interfaced to a liquid delivery system has been constructed. Software has been written to collect and analyze titration data. The prototype had undergone technical testing; laboratory testing with protein, carbohydrate, nucleic acid, and surfactant micelle systems; and end-user evaluation. Experimental protocol have been developed to allow nonspecialist to implement titration experiments with sound velocity as the observable. Based on the experience gained with the prototype, improvements to the design have been made for construction and development of a final precommercialization prototype.
Titration ultrasonic velocimetry is a sensitive method for monitoring molecular associations in solution. It provides unique information on the role of hydration in molecular interactions. The recent invention of a new, mu l volume ultrasonic sensor has made a commercial, easy-to-use, instrument feasible. The Titration Ultrasonic Analyzer (TUSAN) will be the first commercialized ultrasonic instrument capable of monitoring biological samples at practical concentrations and volumes. TUSAN will attract the large community interested in molecular association, including the pharmaceutical and biotechnology industries and academic scientists.
