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.