Low-pressure chromatographic separation and UV/Vis spectrophotometric characterization of the native and desialylated human apo-transferrin
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 purificatio...
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| Format: | Article |
| Language: | English |
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Elsevier
2021-09-01
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| Series: | Heliyon |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405844021021332 |
| _version_ | 1818676867097427968 |
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| author | Tomislav Friganović Antonela Tomašić Tino Šeba Ivan Biruš Robert Kerep Valentina Borko Davor Šakić Mario Gabričević Tin Weitner |
| author_facet | Tomislav Friganović Antonela Tomašić Tino Šeba Ivan Biruš Robert Kerep Valentina Borko Davor Šakić Mario Gabričević Tin Weitner |
| author_sort | Tomislav Friganović |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-17T08:50:17Z |
| format | Article |
| id | doaj.art-1256da0b17dd42de94857db1465e0151 |
| institution | Directory Open Access Journal |
| issn | 2405-8440 |
| language | English |
| last_indexed | 2024-12-17T08:50:17Z |
| publishDate | 2021-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Heliyon |
| spelling | doaj.art-1256da0b17dd42de94857db1465e01512022-12-21T21:56:05ZengElsevierHeliyon2405-84402021-09-0179e08030Low-pressure chromatographic separation and UV/Vis spectrophotometric characterization of the native and desialylated human apo-transferrinTomislav Friganović0Antonela Tomašić1Tino Šeba2Ivan Biruš3Robert Kerep4Valentina Borko5Davor Šakić6Mario Gabričević7Tin Weitner8Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, 10000 Zagreb, CroatiaFaculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, 10000 Zagreb, CroatiaFaculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, 10000 Zagreb, CroatiaFaculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, 10000 Zagreb, CroatiaFaculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, 10000 Zagreb, CroatiaFaculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, 10000 Zagreb, CroatiaFaculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, 10000 Zagreb, CroatiaFaculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, 10000 Zagreb, CroatiaCorresponding author.; Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, 10000 Zagreb, CroatiaLow-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.http://www.sciencedirect.com/science/article/pii/S2405844021021332TransferrinGlycoformsSialic acidChromatographyUV-Vis spectroscopyMolar absorbance |
| spellingShingle | Tomislav Friganović Antonela Tomašić Tino Šeba Ivan Biruš Robert Kerep Valentina Borko Davor Šakić Mario Gabričević Tin Weitner Low-pressure chromatographic separation and UV/Vis spectrophotometric characterization of the native and desialylated human apo-transferrin Heliyon Transferrin Glycoforms Sialic acid Chromatography UV-Vis spectroscopy Molar absorbance |
| title | Low-pressure chromatographic separation and UV/Vis spectrophotometric characterization of the native and desialylated human apo-transferrin |
| title_full | Low-pressure chromatographic separation and UV/Vis spectrophotometric characterization of the native and desialylated human apo-transferrin |
| title_fullStr | Low-pressure chromatographic separation and UV/Vis spectrophotometric characterization of the native and desialylated human apo-transferrin |
| title_full_unstemmed | Low-pressure chromatographic separation and UV/Vis spectrophotometric characterization of the native and desialylated human apo-transferrin |
| title_short | Low-pressure chromatographic separation and UV/Vis spectrophotometric characterization of the native and desialylated human apo-transferrin |
| title_sort | low pressure chromatographic separation and uv vis spectrophotometric characterization of the native and desialylated human apo transferrin |
| topic | Transferrin Glycoforms Sialic acid Chromatography UV-Vis spectroscopy Molar absorbance |
| url | http://www.sciencedirect.com/science/article/pii/S2405844021021332 |
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