Studies on the Role of Sialic Acid in the Physical and Biological Properties of Erythropoietin

Abstract

Crude erythropoietin was subjected to isoelectric focusing over the pH ranges 3–10 and 3–5. All the biological activity was located between pH 3.5 and 4.0. The erythropoietin activity displayed heterogeneity, being divided into three or four peaks within this pH range. Asialoerythropoietin, prepared by acid or enzymic hydrolysis, focused in the pH gradient between pH 5.0 and 6.5 and displayed a similar degree of heterogeneity. Partially desialated erythropoietin focused over a range of intermediate pH values. The large shift in pI on desialation and the demonstration of intermediate partially desialated species suggest a high content of sialic acid in erythropoietin. All desialated species of erythropoietin having isoelectric points greater than 4.0 were without in vivo biological activity. Oxidation of asialoerythropoietin with galactose oxidase completely restored in vivo biological activity.