A simple automatic titrator (suitable for attachment to commercial fluorometers) in which the sample is stirred by a stream of N~ bubbles is described and evaluated_ The chief merits of the device are its flexibility and its suitability for mixing small volumes. Some of the problems encountered while performing fluorescence titrations are described. Appropriate corrections for dilution, optical screening, and other side effects are given. Even after full correction, the overall precision with which a dissociation constant can be measured is __+ 10-20%. Many of the points made are illustrated by a study of the interactions of isoleucine:tRNA ligase with 2-p-toluidinylnaphthalene-6-sulphonate and with its substrates.
A common use of fluorescence in biochemistry is to detect interactions between an emitter and other molecules. The strengths of such interactions are determined by measurement of the appropriate equilibrium constants. These can be calculated from the fluorescence intensities of the emitter in the presence of known concentrations of the second species. Such titrations can also be used to determine the number of binding sites on a macromolecule (using, for example, Scatchard plots) and to measure relative quantum yields. However performance of fluorescence titrations aliquot by aliquot is both tedious and cumulatively inaccurate. We report here a simple device for attachment to commercial fluorometers to permit automatic titrations, and also point out certain pitfalls to be avoided in interpretation of the results.
MATERIALS
L-Isoleucine:tRNA ligase (EC 6.1.1.5) from Escherichia coli Kl2 EM20031 was purified by the method of Baldwin and Berg (1) with slight modifications (2). It migrated as a single band in polyacrylamide gel electrophoresis and had a specific activity in the ATP-PP~ exchange assay (3) of 800 units mg -1. Other reagents were commercial products of high purity. Solutions were prepared in distilled deionised water.
Fluorescence intensities were measured with a Hitachi MPF-2A fluorescence spectrometer modified to have an additional back-off capa-239