Bleomycin-A 2 complexes with poly(dA-dT): A proton nuclear magnetic resonance study of the nonexchangeable hydrogens

Abstract

Binding of bleomycin-A 2 (Bleo-A 2) to poly(dA-dT) in 100 mM sodium phosphate (pH 6.8) in D 2O has been investigated by 1H NMR spectroscopy at 270 and 360 MHz. Significant spectral perturbations were observed only when the nucleic acid was in the duplex state. Of the Bleo-A 2 resonances, the two bithiazole peaks exhibited the largest spectral shiffs and line broadening effects. The high field shift of these resonances was very small near room temperature and reached a maximum of about 0.27 ppm just below the thermal denaturation temperature of the nucleic acid (T m = 60 ± 1°C). The temperature dependence of spectral perturbations may be accounted for by the formation of at least two types of Bleo-A 2 complexes with the polynucleotide. Other perturbed resonances of bleomycin are the S-C H 3 and S-C H 2 of the terminal amine, the C H 2-N resonance of the bithiazole residue, and the C H (CH 3)CO of the methylvaleric acid residue. The significantly perturbed resonances of the nucleic acid originate from the A(H-2), A(H-8), T(H-6) and one of the H-2′ hydrogens. Binding of the C-terminal tripeptide fragment of Bleo-A 2 to poly(dA-dT) is accompanied by selective broadening of the bithiazole group. These experiments have identified potential loci of interaction on the Bleo-A 2 and poly(dA-dT) molecules.