The Buoyant Behavior of Viral and Bacterial DNA in Alkaline CsCl

Abstract

In equilibrium density gradient centrifugation, the banding polymer species is electrically neutral. The banding species for a negative polyelectrolyte with a polyanion P_(n)^(-z)n (where n is the degree of polymerization, and z the titration charge per monomer unit) in a CsCl salt gradient is CS_(zn)P_n. If the ion P_(n)^(-z)n is itself a weak acid, it may be titrated to the state P_(n)^(-(Zn+y)) by CsOH; the banding species is then Cs_(zn+y)P_n. Because of the large mass and high effective "density" of a Cs^+ ion, it is to be expected that the buoyant density in a CsCl gradient of a polymer acid will be increased by such a partial alkaline titration with CsOH. This expectation has been confirmed for polyglutamic acid (where z = 0 at low pH). The guanine and thymine monomer units of DNA are weak acids. The present communication is concerned with the increase in buoyant density of DNA in alkaline CsCl solutions. It is well known that the guanine and thymine protons are more readily titrated in denatured DNA than in native DNA. We find that the buoyant density of denatured DNA and of single strand ϕX-174 DNA gradually increases as the pH of the solution is increased beyond pH 9.8. The density of native DNA is not affected until a critical pH > 11 is reached, where the DNA abruptly denatures and increases in density. Similar increases in buoyant density have been observed independently by Baldwin and Shooter in their studies of 5BU[overbar]-substituted DNA's in alkaline solutions.