Physicochemical and biological characterization of asialoerythropoietin

<jats:p>Various partially or fully desialylated human erythropoietins were obtained by neuraminidase digestion of the hormone, without non‐specific proteolysis and degradation of carbohydrates. Asialoerythropoietin showed a specific activity of 220‐IU/mg protein <jats:italic>in vivo</jats:italic>, although that of the intact erythropoietin was 2.2 × 10<jats:sup>5</jats:sup> IU/mg. A linear relationship was found between the logarithm of the specific activity <jats:italic>in vivo</jats:italic> and the number of sialic acids. The asialoerythropoietin showed a four‐times‐higher specific activity <jats:italic>in vitro</jats:italic> compared with intact erythropoietin using mouse bone marrow cells. It also showed an approximately six‐times‐higher specific activity in a colony‐forming assay for the erythroid colony‐forming unit and the erythroid burst‐forming unit. Partially or fully de‐<jats:italic>N</jats:italic>‐glycosylated erythropoietin derivatives also showed lower <jats:italic>in vivo</jats:italic> activity but higher <jats:italic>in vitro</jats:italic> activity than the intact erythropoietin, dependent on the number of sialic acids. To clarify the reason for the enhanced biological activity of asialoerythropoietin <jats:italic>in vitro</jats:italic>, the binding of intact <jats:sup>125</jats:sup>I‐erythropoietin or <jats:sup>125</jats:sup>I‐asialoerythropoietin to cells containing specific receptors for the hormone was analyzed. <jats:sup>125</jats:sup>I‐asialoerythropoietin bound to spleen cells from anemic mice approximately five times faster than did intact <jats:sup>125</jats:sup>I‐erythropoietin. The amount of <jats:sup>125</jats:sup>I‐asialoerythropoietin internalized by target cells, measured in the absence of NaN<jats:sub>3</jats:sub>, was four times higher than that of intact erythropoietin. These results demonstrate that asialoerythropoietin binds to its receptor faster than the intact form. This may be the main reason for the increased activity of asialoerythropoietin <jats:italic>in vitro</jats:italic>.</jats:p>