DEVELOPMENT OF A DATA ACQUISITION AND
ANALYSIS SYSTEM FOR MEASURING PROTEIN BINDING
CONSTANTS THROUGH ULTRACENTRIFUGATION
Gerald L. Fitzgerald, William J. Cassano, Karl K. Soneson, Frank L. Tobin and Preston Hensley, Smith Kline & French Laboratories, M/S L-331, P.O. Box 1539, King of Prussia, PA 19406-0939
The ultracentrifuge is an important bioanalytical instrument that has long been used for determining the molecular weight and physical characteristics of proteins in solution. More recently, ultracentrifugation has received attention as a method for quantitatively characterizing protein-protein recognition. (Hensley,P.et al.,1986 J.Biol.Chem. 261, 11038-11044). This talk will focus on designing a data collection and analysis system for a Beckman Model E Ultracentrifuge (circa 1950). Data collection must be capable of sampling rates in the range of 2 MHz in order to take advantage of rotor speeds as high as 60,000 rpm. The ultracentrifuge's photomultiplier tube has been directly linked to a CAMAC A/D converter, allowing characterization of the reference and sample intensity peaks. The output from the photomultiplier is collected while peak heights are averaged at each radius until the signal to noise ratio reaches a predetermined level. Emphasis has been placed on statistical noise control over short and long time periods in order to provide consistent signal to noise ratios at different rotor radii and signal levels.
A second aspect of this work has been to model several properties of this data system including the effects of noise, number of data points collected, and A/D precision. These properties have been evaluated with respect to their effect on the calculations for determining molecular weight and concentration. The goal has been to develop an approach such that the data analysis can compensate for bias from the instrument and data acquisition system.