Properties of the binding of copper by bleomycin

Abstract

The glycopeptide, bleomycin, binds metal ions including Cu 2+. It is the copper complex of this material that is isolated from Streptomyces verticillus. Both free ligand and copper complex are excellent antitumor agents in animals. The biochemical and pharmacological relationship between these compounds has not been established. The present study begins an analysis of the chemistry and biochemistry of copper-bleomycin with structural and equilibrium properties of the complex. Potentiometric and fluorometric titrations of bleomycin confirm three acidic groups with p K a values of 7.50, 4.93, and 2.72. The conjugate nitrogen bases of these groups, comprise three of the binding sites for Cu 2+ according to similar titrations of copper-bleomycin. The fourth is a conjugate base of an acid with a very large p K a that cannot be measured by these techniques. The participation of a fourth such group is inferred from both proton release studies of the binding of metal and ligand above pH 8 and from several studies of the thermodynamic stability of copper bleomycin. At low pH binding of copper to bleomycin occurs in two steps, as observed by several independent techniques which monitor either the metal or the ligand. Log stability constants for the reactions Cu 2+ + H k Blm ⇌ CuH k-n Blm + nH + and CuH k-n Blm ⇌ CuH k-n-r Blm + rH + are 1.32 and −4.31, respectively, with n of 2.21 in the first equation and r of 2.07 in the second equation. The derived logarithm of the pH independent stability constant for copper bleomycin multiplied by the protonation constant for the unknown fourth ligand in the binding site is 12.16. This agrees closely with values obtained from measurements of conditional formation constants. One of the groups which binds in the second reaction is the substituted pyrimidine.