1st YEAR – 2018

WP1 Installation of the research group (Weitner, Šakić, Biruš, Friganović)

In the initial phase, the required workspace and laboratory renovation will take place. The research team will be expanded for subsequent phases by employing the PhD student who bill be accommodated in a dedicated working room with internet access. All necessary chemicals and materials for the initial phase of research will be purchased and additional activities of the initial research phase will take place, including literature overview, establishment and verification of methods and protocols (chromatographic procedures, protein purification/concentration, spectroscopic procedures, including UV/Vis spectroscopy, spectrofluorometry, multimode microplate reader).

Task 1.1: Laboratory and workspace adaptation (installations, furniture) - COMPLETED

Task 1.2: Procurement of necessary chemicals and materials, including new equipment, access to the Lexicomp online database, small lab equipment, consumables etc. - COMPLETED

Task 1.3: Employment of the PhD student - COMPLETED

Task 1.4: Establishment and verification of methods and protocols- COMPLETED

Task 1.5: Dissemination activities (FARMEBS, web page) - COMPLETED

HRZZ UIP-2017-05-9537

Home        Team        Research        Collaborations        Activities        Publications

2nd YEAR – 2019

WP2 Preparation and identification of transferrin glycoforms
(Weitner, Šakić, Biruš, Friganović, Borko)

Preliminary data will be collected and evaluated (glycan analysis of native transferrin) and specific training for experimental design and data evaluation of the preparation and identification of transferrin glycoforms will be performed (mass spectroscopy). The optimal protocols for the identification and isolation of transferrin glycoforms, detailed glycan structure analysis and determination of the spectral characteristics of the proteins will be established. These protocols will be used for the preparation of additional quantities of protein samples for later phases.

Task 2.1: Specialization training for experimental design and data evaluation
                (Duke University, Friedrich-Alexander University) - COMPLETED

Task 2.2: Collection and evaluation of preliminary data
                (glycan analysis of native transferrin) - COMPLETED

Task 2.3: Identification and isolation of transferrin glycoforms in sufficient amounts for use in iron binding experiments (additionally repeated as necessary) - COMPLETED

Task 2.4: Determination of the detailed glycan structures of the isolated glycoforms - COMPLETED

Task 2.5: Determination of the spectral characteristics of the isolated glycoforms - COMPLETED

Task 2.6: Dissemination activities (FARMEBS, conferences) - COMPLETED


3nd YEAR – 2020

WP3 Determination of the iron-binding equilibrium constants for transferrin glycoforms (Weitner, Šakić, Biruš, PhD student)

The iron-binding equilibrium constants will be determined by spectrophotometric and spectrofluorometric titrations with FeNTA. In order to increase throughput and minimize sample waste we will use the multimode microplate reader and microvolume plate. We will use an automated plate washer to prevent carryover and facilitate microplate loading. A model for the calculation of both stoichiometric association constants will be formulated, accounting for all equilibria in the solution. A manuscript of the publication on the reduction potentials of iron bound to transferrin glycoforms will be written.

Task 3.1: UV-Vis spectrophotometric measurement of iron binding equilibrium at pH = 7.4 in the presence of phosphate, citrate, NTA, oxalate and carbonate as exogenous ions – COMPLETED

Task 3.2: UV-Vis spectrophotometric measurement of iron binding equilibrium at pH = 5.8 in the presence of phosphate, citrate, NTA, oxalate and carbonate as exogenous ions – COMPLETED

Task 3.3: Spectrofluorometric measurement of iron binding equilibrium at pH = 7.4 in the presence of phosphate, citrate, NTA, oxalate and carbonate as exogenous ions – IN PROGRESS

Task 3.4: Spectrofluorometric measurement of iron binding equilibrium at pH = 5.8 in the presence of phosphate, citrate, NTA, oxalate and carbonate as exogenous ions – IN PROGRESS

Task 3.5: Calculation of stoichiometric association constants for the binding of iron to transferrin glycoforms – IN PROGRESS


4th YEAR – 2021

WP4 Determination of the reduction potential of iron bound to transferrin glycoforms (Weitner, Biruš, Friganović, Borko)

The reduction potentials will be determined by a spectroelectrochemical (SEC) method in as SEC flow cell with disposable screen-printed indium-tin oxide electrodes. The dependence of iron binding sites heterogeneity on the glycosylation pattern will be measured by EPR spectroscopy. Specific training for experimental design and data evaluation for quantum-chemical modeling of Fe(III) and Fe(II) complexes in the protein binding sites will be performed and subsequently the corresponding binding energies, HOMO/LUMO/SOMO orbitals and geometry will be determined. A manuscript of the publication on stoichiometric association constants for the binding of iron to transferrin glycoforms will be written.

Task 4.3: Spectroelectrochemical determination of Fe-saturated diferric (Fe2Tf), monoferric C lobe (FeCTf), and monoferric N-lobe (FeNTf) reduction potentials at pH = 7.4 in the presence of carbonate, oxalate, citrate, phosphate – IN PROGRESS

Task 4.4: Spectroelectrochemical determination of Fe-saturated diferric (Fe2Tf), monoferric C lobe (FeCTf), and monoferric N-lobe (FeNTf) reduction potentials at pH = 5.8 in the presence of carbonate, oxalate, citrate, phosphate  – IN PROGRESS

Task 4.5: Quantum-chemical modeling of binding energies, HOMO/LUMO/SOMO orbitals and geometry of Fe(III) and Fe(II) complexes in the protein binding sites in the presence of carbonate, oxalate, citrate, phosphate  – IN PROGRESS


WP5 Determination of the kinetics and mechanism of iron binding to transferrin glycoforms (Weitner, Biruš, Friganović, Borko)

The iron-binding kinetics of the reaction of apotransferrin and FeNTA will be followed by spectrophotometry and spectrofluorometry. In order to increase throughput and minimize sample waste we will use the multimode microplate reader and microvolume plate. For faster kinetic steps we will use the rapid-scan stopped-flow apparatus. We will use an automated plate washer to prevent carryover and facilitate microplate loading. A kinetic model that accounts for all observed kinetic steps will be constructed.

Task 5.4: Spectrofluorometric measurement of iron binding kinetics at pH = 7.4 in the presence of phosphate, citrate, NTA, oxalate and carbonate as exogenous ions – IN PREPARATION

Task 5.5: Spectrofluorometric measurement of iron binding kinetics at pH = 5.8 in the presence of phosphate, citrate, NTA, oxalate and carbonate as exogenous ions – IN PREPARATION

Task 5.6: Kinetic modeling of iron binding to transferrin glycoforms – IN PREPARATION