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

3rd YEAR – 2020

WP3 Determination of the iron-binding equilibrium constants for transferrin glycoforms (Weitner, Biruš, Friganović, Borko)

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 - IN PROGRESS

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 - IN PROGRESS