A Practical Method for Preparing Copper-Free Bleomycin A₂

Abstract

A solution of 3.0 g of copper-containing bleomycin A₂ hydrochloride (930 meg potency/mg. 4.28% copper content) in 100 ml of distilled water was passed through a column packed with 1 liter of Amberlite XAD-2 in distilled water to effect adsorption of bleomycin A₂.Then 3 liters of a 5% aqueous solution of Na-EDTA was applied to the column to remove Cu-EDTA. The column was washed with 1 liter of a 5% sodium chloride solution and then with 2 liters of distilled water. Finally, 1.5 liters of a mixture (1: 1) of 2.5 mM hydrochloric acid and acetone was allowed to flow through the column to elute bleomycin A₂.The latter half of the eluate, 700 ml in volume, was collected and concentrated under diminished pressure. The residue was subjected to reprecipitation from water-acetone, and dried to obtain 2.8 g of copper-free bleomycin A₂ hydrochloride (945 meg potency/mg; 0.0083% copper content) in the form of a pale yellow powder; yield 93.3%, absorbance (aqueous solution) at 292 nm $E_{1{\text{cm}}}^{1\% } = 103.$