The sensitivity of the bleomycin assay for loosely-bound iron depends on the concentration of bleomycin and ascorbic acid and the pH of the reaction. The non-haem-iron proteins transferrin, conalbumin and ferritin release iron at an acid pH value, whereas the haem-iron proteins release iron more readily at an alkaline pH. In addition, haem proteins are liable to release iron when peroxides are present. Organic peroxides and hydrogen peroxide can be produced during the bleomycin reaction leading to iron release from haem proteins. However, this can be prevented from reacting with bleomycin by adding zinc ions to the reaction following addition of the sample. Iron already bound to bleomycin is not displaced by zinc whereas zinc bound to bleomycin is not displaced by iron allowing 'free' and 'released' iron to be discriminated.