Dresden 2003 – wissenschaftliches Programm

Bereiche | Tage | Auswahl | Suche | Downloads | Hilfe

CPP: Chemische Physik und Polymerphysik

CPP 21: POSTER B

CPP 21.8: Poster

Dienstag, 25. März 2003, 19:00–21:00, ZEU/250

Kinetic and Thermodynamic Analysis of ssDNA–RPA Interactions with SPR and FCS — •Schubert F.¹, Zettl H.¹, Krauss G.², and Krausch G.¹ — ¹Physikalische Chemie II, Universität Bayreuth, 95447 Bayreuth — ²Laboratorium für Biochemie, Universität Bayreuth, 95447 Bayreuth

Eukaryotic replication protein A (RPA) is a single stranded DNA-binding protein with multiple functions in DNA replication, repair and genetic recombination. We report a kinetic and thermodynamic analysis of ssDNA–RPA interactions using surface plasmon resonance (SPR) and fluorescence correlation spectroscopy (FCS) at various temperatures. The analysis includes the determination of the association and dissocitaion rate constants, the equilibrium constant and the Gibbs free energy. By performing the reaction at different temperatures, it is possible to extract thermodynamic information like reaction enthalpy and entropy about the system using van’t Hoff analysis.

The two applied methods gave different values for the Gibbs free energy but nearly the same value for the reaction enthalpy of ssDNA–RPA complex formation. The Gibbs free energy was determined by SPR and FCS to be -62.2 kJ/mol and -54.7 kJ/mol, respectively. The values for the reaction enthalpy are -64.2 kJ/mol and -66.5 kJ/mol. Thus the difference in Gibbs free energy measured by the two methods is due to different reaction entropies. SPR is a two dimensional method due to the immobilized DNA, FCS is able to measure the diffusion of molecules in three dimension. Thus the reaction entropy for surface plasmon resonance is lower than for fluorescence correlation spectroscopy.