Chromatographic identification of added iron in casein and casein components

The objective of this study was to determine the distribution of added radioiron in milk protein and casein components of cow's milk fractionated by the combination of Sephadex G-150 gel filtration and DEAE-cellulose chromatography. Labelled ⁵⁹FeCl₃ was added directly to raw milk at the concentration of 0.02 and 10 ppm isotope and carrier, respectively, and held overnight at 4°C. Five ml of the skim milk was first chromatographed on Sephadex G-150 column and fractionated into casein, whey protein, and nonprotein materials. The casein fraction was chromatographed on a DEAE-cellulose column and separated into its components, α_s, β-, and κ-caseins. Radioactivity was determined first in skim milk and later in casein, whey protein, and casein components. The data indicated that most of the added iron was associated with skim milk. Sephadex G-150 gel permeation of skim milk showed that the casein was associated with more than 85 percent of the activity in the skim milk. DEAE-cellulose chromatography of casein showed that the added iron was bound mostly to α_s-casein and β-casein. Surprisingly, very little radioactivity, about 5 percent, was bound to κ-casein.