o:37113 A highly optimized protocol for the isolation and purification of transferrin from human serum en Different methods for the isolation and purification of human transferrin (Tf) from human plasma and se rum have been described in the literature. However, none provide a highly pure final product in a time-ef ficient manner, whereas the method described in this work accomplishes both. It consists of two separate steps—a sample preparation and an anion-exchange chromatography step. Our initial methodology used rivanol for protein precipitation in the first step and activated charcoal for the removal of excess rivanol from the remaining supernate. This provided Tf of >99% purity while retaining Tf function, analyzed by immunofluorescent staining for transferrin-transferrin receptor 1 in teraction and ferrozine method for iron binding capacity, and structure, confirmed by recording and comparing fluorescent spectra of purified Tf and commercially available pure Tf. However, the method produced a relatively low Tf yield. The initial method was then modified to use potassium bromide for excess rivanol removal, increasing yield by approximately 82% compared to the initial protocol. We then tested if saturating Tf with iron(III) ions before precipitation with rivanol would increase yield. This increased yield by a further 20% compared to potassium bromide rivanol removal, and a final 119% compared to the initially proposed method. The improved method was additionally evaluated for the purification of Tf from the serum of patients with different iron-associated pathologies, showing its applicability on different types of samples. transferrin, anion-exchange chromatography, protein purification 1552100 978-86-7220-136-9 2025-11-21T12:31:54.746Z 44 yes 46 Danilo Četić University of Belgrade, Institute for the Application of Nuclear Energy, Serbia master biolog 0000-0001-9705-3237 Goran Miljuš University of Belgrade, Institute for the Application of Nuclear Energy INEP, Serbia doktor biohemijskih nauka person 0000-0001-6417-8711 Jovana Stevanović University of Belgrade, Institute for the Application of Nuclear Energy INEP, Serbia master biolog person 0000-0002-3619-8472 Zorana Dobrijević University of Belgrade, Institute for the Application of Nuclear Energy INEP, Serbia doktor biohemijskih nauka person 0000-0002-0898-5401 Miloš Šunderić University of Belgrade, Institute for the Application of Nuclear Energy INEP, Serbia doktor biohemijskih nauka person 0000-0002-0940-9481 Olgica Nedić Institute for the Application of Nuclear Energy INEP, University of Belgrade, Serbia doktor biohemijskih nauka person 0000-0003-2042-0056 Nikola Gligorijević University of Belgrade, Institute for the Application of Nuclear Energy INEP, Serbia person Aleksandra Vilotić Institute for the Application of Nuclear Energy INEP, University of Belgrade, Serbia doktor bioloških nauka person 0000-0002-0898-5401 Valentina Borko Department of Biochemistry and Molecular Biology, University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, Croatia person Mario Gabričević Department of General and Inorganic Chemistry, University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, Croatia person Tin Weitner Department of General and Inorganic Chemistry, University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, Croatia person Ana Penezić Institute for the Application of Nuclear Energy INEP, University of Belgrade, Serbia doktor biohemijskih nauka person 0000-0002-2539-4507 application/pdf 625323 https://phaidrabg.bg.ac.rs/o:37113 no yes 18 70 92000004 11A29 A highly optimized protocol for the isolation and purification of transferrin from human serum 135 136 FEBS3+ Meeting "Advances in Molecular Biosciences: From Genes to Personalized Therapies" – Book of Abstracts Beograd, Srbija Hemijski fakultet, Srpsko biohemijsko društvo 2025