Low-pressure chromatographic separation and UV/Vis spectrophotometric characterization of the native and desialylated human apo-transferrin

Friganović, Tomislav; Tomašić, Antonela; Šeba, Tino; Biruš, Ivan; Kerep, Robert; Borko, Valentina; Šakić, Davor; Gabričević, Mario; Weitner, Tin

Low-pressure pH gradient ion exchange separation provides a fast, simple and cost-effective method for preparative purification of native and desialylated apo-transferrin. The method enables easy monitoring of the extent of the desialylation reaction and also the efficient separation and purification of protein fractions after desialylation. The N-glycan analysis shows that the modified desialylation protocol successfully reduces the content of the sialylated fractions relative to the native apo-transferrin. In the optimized protocol, the desialylation capacity is increased by 150 %, compared to the original protocol provided by the manufacturer. The molar absorption coefficients in the near-UV region for the native and desialylated apo-transferrin differ by several percent, suggesting a subtle dependence of the glycoprotein absorbance on the variable sialic acid content. The method can easily be modified for other glycoproteins and is particularly appropriate for quick testing of sialic acid content in the protein glycosylation patterns prior to further verification by mass spectrometry.