Effect of hydrostatic pressure upon ethidium bromide association with transfer ribonucleic acid.

Abstract

The binding of ethidium bromide to yeast phenylalanine-specific transfer ribonucleic acid (tRNAPhe) has been investigated in the pressure range from 1 atm to 9 kbar in the presence of 100 mM sodium chloride and 10 mM magnesium chloride, pH 7.7. One high-affinity binding site for ethidium is present, with a dissociation constant of 2.4 X 10(-6) M at 1 atm and 22 degrees C. Binding to this site is enhanced with increasing pressure, the dissociation constant reaching 2.9 X 10(-7) M at 2 kbar. Pressure also promotes the binding of ethidium to lower affinity sites of tRNAPhe. The standard volume change upon complex formation is found to be 25.6 +/- mL/mol for the first ethidium bound. If sodium is replaced by lithium in the buffer, the standard volume change is 23.3 +/- 0.5 mL/mol. We conclude that decrease of the electrostatic repulsion in the negatively charged tRNAPhe by binding of the positively charged ethidium is the main cause of the relatively large volume decrease upon complex formation. The electrostatic repulsion that must be present in this case, as well as in other nucleic acids, implies that intercalating binding sites are of the "soft" type as previously defined [Torgerson, P.M., Drickamer, H.G., & Weber, G. (1979) Biochemistry 18, 3079]. Model studies by others of the binding site characteristics are in agreement with this concept.