Ultracentrifugal analysis of protein-nucleic acid interactions using multi-wavelength scans
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 p 0 for the interaction.
Key wordsUltracentrifugal analysis proteins nucleic acids molecular interactions
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- 5.Wissman A, Hillen W (1991) In: Sauer, RT (ed) Methods in Enzymology, Vol 208: Protein-DNA Interaction. Academic Press, New York, pp 365–379Google Scholar
- 7.Dixon, WJ, Hayes JJ, Levin JR, Weidner MF, Dombroski BA, Tullius TD (1991) In: Sauer, RT (ed) Methods in Enzymology, Vol. 208: Protein-DNA Interaction. Academic Press, New York, pp 380–413Google Scholar
- 8.Dervan P (1991) IN: Sauer RT (ed) Methods in Enzymology, Vol. 208: Protein-DNA Interaction. Academic Press, New York, pp 497–515Google Scholar
- 9.Koblan KS, Bain DL, Beckett D, Shea MA, Ackers G (1992) In: Brand L, Johnson M (eds) Methods in Enzymology, Vol. 210: Numerical Computer Methods. Academic Press, New York, pp 497–515Google Scholar
- 13.Lewis MS, Kim S-J, Kumar A, Wilson SH (1992) Biophys J 61:A489Google Scholar
- 14.Schmidt B, Riesner D (1992) In: Harding SE, Rowe AJ, Horton JC (eds) Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, Cambridge, pp 176–207Google Scholar
- 15.Lewis MS (1991) Biochemistry 30:11716–11719Google Scholar
- 16.Strang G (1986) In: Intrduction to Applied Mathematics. Wellesley-Cambridge Press, Wellesley, MA, pp 138–139Google Scholar
- 17.Durchschlag H (1986) In: Hinz H-J (ed) Thermodynamic Data for Biochemistry and Biotechnology. Springer-Verlag, New York, pp 46–50Google Scholar
- 20.Jansen DE, Kelly RC, von Hippel PH (1976) J Biol Chem 251:7215–7228Google Scholar
- 19.Pratt WK (1978) In: Digital Image Processing. John Wiley and Sons, New York, pp 206–211Google Scholar