Odjeljivanje formi α-1 kiselog glikoproteina niskotlačnom kromatografijom anionske izmjene

master's thesis

• Title (english): Separation of α-1 glycoprotein forms using low pressure anion exchange chromatography
• Author: Jelena Radovčić
• Mentor: Mario Gabričević (mentor)
• Committee member: Mario Gabričević (predsjednik povjerenstva)
• Committee member: Tin Weitner (član povjerenstva)
• Committee member: Davor Šakić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of general and inorganic chemistry); Zagreb
• Defense date and country: 2022-10-03, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Cilj ovog rada bio je odijeliti frakcije AGP-a različitih izoelektričnih točaka anionsko izmjenjivačkom kromatografijom. AGP je protein plazme čije su različite forme zabilježene kako u peptidnom tako i u glikanskom dijelu. Različite glikoforme i genske varijante AGP-a pokazuju različita fizikalno-kemijska svojstva, a jedno od tih svojstava je i pI molekule što otvara mogućnost analize AGP-a kromatofokusiranjem. Korištena metoda, kromatofokusiranje, bazirana je na stvaranju ionskih veza između nabijenih grupa biomolekula i ionsko-izmjenjivačkog gela/nosača koji nosi suprotan naboj. Promjenom pH otopine dolazi do promjene u naboju biomolekule te u njezinoj eluaciji s kolone. Zbog različitih izoelektričnih točaka formi, različit pH otopine će uzrokovati promjenu naboja u biomolekuli te forme što se očituje različitim retencijskim vremenom. Nativna (sijalinizirana) forma AGP-a je analizirana tri puta anionsko izmjenjivačkom kromatografijom, koristeći jednu, tri i pet kolona dok je AGP koji je prethodno desijaliniziran, analiziran anionsko izmjenjivačkom kromatografijom koristeći pet kolona. Rezultati analize su grafički prikazani koristeći Excel program, prikazujući ovisnost pH otopine i apsorbancije uzroka o volumenu otopine. Iz grafičkog prikaza je vidljivo da su korištenjem metode nepotpuno odijeljene dvije frakcije AGP-a različitih izoelektričnih točaka dok se povećanjem korištenih kolona, povećala razlučivost metode.

Abstract (english)

The goal of this work was to separate fractions of AGP by using anion exchange chromatography. AGP is a plasma protein with different forms in its peptide and glycan portion of molecule. Different glycoforms and genetic variants of AGP have different physico-chemical properties. pI as one of those properties enables separation of AGP forms by using the chromatofocusing technique. Chromatofocusing is based on the formation of ionic bonds between charged groups of the biomolecule and ion-exchange gel/carrier carrying opposite charge. Changing the pH of the solution, causes the change of the molecule charge and its elution of the column. Due to the different isoelectric points of the forms, different values of pH in the solution change charge of the molecule, which leads to forms having different retention times at column. Native (sialylated) form of AGP was analized by using one, three or five columns in a row of anion exchange chromatography while AGP that was previously desialylated was analyzed using anion exchange chromatography only with five columns in a row. Results show the relation between pH or absorbance of the sample and volume of the solution. It can be concluded that two forms of AGP with different isoelectric points were separated, but not completely. Using a higher number of columns increased separation.

• Keywords: α-1 kiseli glikoprotein; genske varijante; glikoforme; kromatografija anionske izmjene; kromatofokusiranje
• Keywords (english): α-1 glycoprotein; genetic variants; glycoforms; anion exchange chromatography; chromatofocusing
• Language: croatian
• URN:NBN: urn:nbn:hr:163:231876
• Study programme: Title: Pharmacy; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra farmacije (magistar/magistra farmacije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2022-11-03 15:41:01