Ultracentrifugal analysis of protein-nucleic acid interactions using multi-wavelength scans

Abstract

The use of analytical ultracentrifugation for the investigation of the interactions of proteins and nucleic acids has been studied by computer simulartions and experimentally in the Beckman XL-A analytical ultracentrifuge. The technique involves obtaining absorbency data at wavelengths in the range of 230 to 246 nm as well as at 260 and 280 nm for the protein solution, the nucleic acid solution, and a solution of these reactants and their complex. The data from the protein and nucleic acid solutions permits calculation of the molar extinction coefficients of these reactants as functions of wavelength which then can be used to construct an extinction coefficient matrix. The data from the solution of reactants and complex permits constructing an absorbency matrix which is a function of radius and wavelength. These matrices may then be used to obtain a data matrix with radial positions in the first column and molar concentrations of protein plus complex and nucleic acid plus complex in the second and third columns. This data matrix can then be analyzed by mathematical modeling to obtain the value of the natural logarithm of the molar equilibrium constant. The computer simulation study demonstrates that the generating value of ln K is recovered with very little error in spite of the presence of substantial random error added to the absorbency data; the experimental study demonstrates that the method can be applied with equal facility to data obtained with the XL-A ultracentrifuge. The temperature dependence of these values of ln K can then be used to obtain values of ΔG ⁰, ΔH ⁰, ΔS ⁰ and ΔC ⁰_p for the interaction.

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