A stopped-flow H-D exchange kinetic study of spermine-polynucleotide interactions.

Abstract

The rates of H-D exchange for imino and amino protons in adenosine, calf thymus DNA, poly (dA-dT), poly(dG-dC), and poly (dG-me5dC) were determined using stopped flow kinetic methods in the presence of various concentrations of Tris, imidazole, Mg2+, and spermine in citrate buffer (pH 7, 25 degrees C). CD spectroscopic studies showed that all polynucleotides always remain in the B-form under these conditions. An increase in the concentration of Tris and imidazole from 5 mu M to 20 mM caused an increase in the rates of exchange of both fast-exchanging imino and slow-exchanging amino protons. The limiting rates of exchange at infinite concentrations of catalysts were found to be different for fast (31-57 sec-1) and slow (1-2 sec-1) exchanging protons. These results indicate that imino and amino protons of B-DNA exchange asymmetrically from two different open states as observed for Z-DNA. An increase in the concentration of spermine from a ratio of 1:50 to 1:2 of positive charge/phosphate decreased the rate of exchange of imino protons of calf-thymus DNA, poly(dG-dC), and poly(dG-me5dC), but increased the rate of exchange of the imino protons of poly(dA-dT) without affecting the exchange rate of the amino protons of any of the polynucleotides. These results are interpreted in terms of possible spermine-induced change of conformations of oligonucleotides of specific sequence that has been suggested by theoretical model building studies.