Interference of melanin in protein determination

Using several assay methods, synthetic eumelanin prepared by autooxidation of L-beta-(3,4-dihydroxyphenyl)alanine and a natural melanin isolated from dog hair melanosomes were tested in model experiments to assess their possible interference in protein determination. The degree of interference was assessed by comparing the data obtained with the melanin samples with those derived from measurements of bovine serum albumin. In the common biuret and Lowry methods melanin interferes by falsely increasing the values obtained; the addition of Folin reagent only after melanin removal, as suggested by Doezema, decreased but did not eliminate melanin interference. Methods working at acid pH such as those according to Salo and Honkavaara with Ponceau S or Sedmak and Grossberg or Spector using Coomassie blue G-250 proved much better. Although melanins adsorbed a small amount of dye from the reaction systems in these procedures, their sensitivity to proteins makes the melanin interference negligible. Such procedures can therefore be recommended for protein determination in the presence of melanin.