112
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.