Experimental Design and Polyelectrolyte Effects on Ligand Binding to Nucleic Acids. An Undergraduate Biochemistry Lab

• Matthew A. Fisher ^[1] ; Danielle Johnston ^[1] ; Daniel A. Ritt ^[1]
  1. [1] Saint Vincent College

     Saint Vincent College

     Borough of Latrobe, Estados Unidos

     
• Localización: Journal of chemical education, ISSN 0021-9584, Vol. 79, Nº 3, 2002, pág. 374
• Idioma: inglés
• Texto completo no disponible (Saber más ...)
• Resumen

  • We have developed an experiment that utilizes the salt dependence of spermine binding to DNA as a means to introduce experimental design concepts and polyelectrolyte effects in the undergraduate biochemistry lab. The experiment is easily completed in two, three-hour lab periods. Spermine, a multivalent polyamine, competes with ethidium bromide for binding to DNA. Consequently the decrease in the observed ethidium binding constant can be used to determine the equilibrium constant for spermine binding. Binding constants are determined at various concentrations of Na+ and Mg2+ cations in a simple full factorial design. Since cations accumulate near the surface of DNA owing to the polyelectrolyte nature of the nucleic acid, these cations must be displaced by spermine binding. Competition between monovalent and divalent cations for accumulation near the DNA surface results in the two variables being linked; the presence of Mg2+ in the reaction buffer alters how the spermine binding constant changes when the Na+ concentration is altered. Completion of this experiment not only gives students experience with the construction of Scatchard plots but also provides an introduction to experimental design and the importance of polyelectrolyte behavior in nucleic acid chemistry.