HUMAN TRANSFERRIN, ASIALOTRANSFERRIN AND THE INTERMEDIATE FORMS

Six protein fractions were isolated from mixtures of neuraminidase-treated and untreated samples of human transferrin type C (TfC) in the 2Fe-form by chromatography on DEAE-cellulose. The first 2Fe-TfC peak (designated TfC-1–6) to desorb from the column did not contain sialic acid; The second (TfC-2–6) and third (TfC-3–6) peaks were intermediates between asialotransferrin and the least acidic component of native 2Fe-TfC; The fourth, fifth and sixth (TfC-4–6, -5–6 and -6-6) peaks were identical with the three components described earlier for native 2Fe-TfC. Ratios of N-acetylneuraminic acid/galactose contents in all peaks were determined by gas-liquid chromatography of the glycopeptides. Further heterogeneity in the carbohydrate composition of TfC-1–6 was demonstrated with the aid of an affinity medium which was obtained by immobilising the asialoglycoprotein-binding lectin from rabbit liver on Sepharose. This medium separated TfC-1–6 into a retarded (‘low affinity’) and a retained (‘high affinity’) component, the ratios of which in TfC-1–6 samples prepared from three individual donors were similar (83–84% and 16–17% respectively). Column chromatography on Sepharose-concanavalin A (S-con A) was used to identify the position in the normal DEAE-chromatogram of those 2Fe-TfC molecules which, in the desialylated state, give rise to the high-affinity component in TfC-1–6. S-con A divided native 2Fe-TfC into two components: one that passed through the column with 0.1M NaCl and another which could only be eluted by α-methyl-D-glucoside. After mixing the former component with unfractionated TfC, the electrophoretic band corresponding to TfC-6-6 was markedly enriched, and when partially purified TfC-6-6 was chromatographed on S-con A, a large portion of the load failed to specifically bind to the lectin. In contrast, TfC-5–6 was nearly quantitatively retained by S-con A. Resolution of TfC-1–6 on S-con A was similar to that of native 2Fe-TfC. From animal studies the protein not retained by S-con A was identified as high-affinity TfC-1–6, whereas the retained portion was of the low-affinity type. These observations suggest that those 2Fe-TfC molecules which, after desialylation, show high affinity for the hepatic lectin probably correspond to TfC-6-6 in the native state. Complete desialylation of TfC in a single incubation with neuraminidase appears virtually impossible but homogeneous preparation of TfC-1–6 can be obtained by chromatography of the incubation mixture on DEAE-cellulose.