Ispitivanje razlike u hidrofobnosti nativnog i desijaliziranog transferina reakcijom sa 8-anilinonaftalensulfonskom kiselinom

master's thesis

• Title (english): Analysis of the difference in hydrophobicity of native and desialylated transferrin by reaction with 8-anilinonapthalenesulfonic acid
• Author: Tamara Atelj
• Mentor: Tin Weitner (mentor)
• Committee member: Tin Weitner (predsjednik povjerenstva)
• Committee member: Mario Gabričević (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: 2022-09-09, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

U sklopu ovoga diplomskog rada proučavan je utjecaj vezanja hidrofobne probe, 8-anilino-1-naftalensulfonske kiseline, na fluorescenciju humanog nativnog apo-transferina i desijaliniziranog transferina, kao i stehiometrija vezanja kiseline na nativni protein. Ispitivanje hidrofobnosti provedeno je u fosfatnom puferu koncentracije 50 mM, dok je analiza stehiometrije vezanja hidrofobne probe na ljudski apo-transferin provedena u fosfatnom puferu koncentracije 200 mM, a ispitivanja provedena su pri neutralnoj vrijednosti pH i temperaturi od 25°C. Tehnike koje su korištene u eksperimentalnom dijelu rada su UV-Vis spektrofotometrija i fluorescencijska spektroskopija. Rezultati su obrađeni u programu Microsoft Excel. Dodatak ANS otopini ljudskog nativnog apo-transferina uzrokovao je primjetan porast intenziteta fluorescencije, što ukazuje na to da se proba veže za izloženi džep na površini polipeptidnog lanca. Analizom otopine ANS i desijaliziranog ljudskog apo-transferina nije primjećen porast intenziteta fluorescencije, što znači da nije došlo do hidrofobnih interakcija kao u slučaju nativnog apo-transferina. Ispitivanje stehiometrije vezanja ANS i ljudskog nativnog apo-transferina korištenjem Jobove metode ukazuje na to da se proba i protein vežu u omjeru 1:1 i 2:1.

Abstract (english)

As part of this thesis, the influence of binding of the hydrophobic probe, 8-anilino-1-naphthalenesulfonic acid, on the fluorescence of human native apo-transferrin and desialylated transferrin, as well as the stoichiometry of ANS binding to the native protein, were studied. The hydrophobicity test was performed in a phosphate buffer with a concentration of 50 mM, while the analysis of the stoichiometry of binding of the hydrophobic probe to human apo-transferrin was performed in a phosphate buffer with a concentration of 200 mM, and both tests were performed at a neutral value pH and temperature of 25°C. The techniques used in the experimental part of the thesis are UV-Vis spectrophotometry and fluorescence spectroscopy. The results were processed in Microsoft Excel. Addition of ANS to a solution of human native apo-transferrin caused a marked increase in fluorescence intensity, indicating that the probe binds to an exposed pocket on the surface of the polypeptide chain. Analysis of the solution containing the probe and desialylated human apo-transferrin did not show an increase in fluorescence intensity, which indicates that no hydrophobic interactions occurred as was the case with native apo-transferrin solution. Analysis of the stoichiometry of binding of ANS and human native apo-transferrin using Job's plot indicates that the hydrophobic probe and protein bind in a ratio of 1:1 and 2:1.

• Keywords: transferin; 8-anilinonaftalensulfonska kiselina; fluorescencija; stehiometrija vezanja; Jobova metoda
• Keywords (english): transferrin; 8-anilinonapthalenesulfonic acid; fluorescence; stoichiometry of binding; Job's plot
• Language: croatian
• URN:NBN: urn:nbn:hr:163:404231
• 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: 2022-09-09 13:04:01