Vpliv temperature na ločbo bioloških makromolekul

Bavčar, Mojca (author), Pompe, Matevž (mentor)

Reverznofazna kromatografija poleg analize proteinov omogoča tudi vpogled v mehanizem interakcije proteina s hidrofobno površino in ali ta vključuje procese, kot je sprememba njegove konformacije. Za štiri proteine (insulin, citokrom c, lizocim in BSA) sem preučila vpliv temperature na retencijsko obnašanje na reverznofazni koloni C18. Analiza insulina v šestih kombinacijah organske (ACN, MeOH, EtOH) in vodne faze (0.1 % TFA, 0.1 M KCl) je pokazala, da ima izbira organske komponente mobilne faze največji vpliv na temperaturno odvisnost retencijskega časa. Primerjala sem retencijsko obnašanje insulina in citokroma c v dveh mobilnih fazah, 0.1 % TFA-ACN (70:30) in 0.1 % TFA-MeOH (44:56), pri konstantnem pretoku in tlaku. Van't Hoffovi grafi in iz njih izračunane termodinamske količine kažejo, da se v molekuli insulina v TFA-ACN pri ~ 53 °C začnejo večje konformacijske spremembe, medtem ko do teh v TFA-MeOH prihaja že pri nižjih temperaturah. Podobno so večje konformacijske spremembe citokroma c v TFA-ACN prisotne pri nižjih temperaturah, v TFA-MeOH pa do teh ne prihaja. Lizocim in BSA sem analizirala z gradientno metodo 0.1 % TFA-ACN. Trendi retencijskih časov kažejo, da tudi v teh dveh proteinih prihaja do konformacijskih sprememb.

• Language: Slovenian
• Keywords: kromatografija proteinov, nelinearni van't Hoffov graf, insulin, citokrom, lizocim, BSA
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2021
• PID: 20.500.12556/RUL-128963
• COBISS.SI-ID: 75435011
• Publication date in RUL: 19.08.2021

Secondary language

• Language: English
• Title: Influence of temperature on separation of biological macromolecules

Abstract

In addition to protein analysis, reversed-phase chromatography also provides insight into mechanism of interaction between protein and hydrophobic surface and whether it includes processes such as change of protein conformation. I studied influence of temperature on retention behaviour of four proteins (insulin, cytochrome c, lysozyme and BSA) on reversed-phase C18 column. Analysis of insulin in six combinations of organic (ACN, MeOH, EtOH) and aqueous phases (0.1 % TFA, 0.1 M KCl) indicates that choice of organic component of mobile phase has greatest influence on temperature dependence of retention time. I compared retention behaviour of insulin and cytochrome c in two mobile phases, 0.1 % TFA-ACN (70:30) and 0.1 % TFA-MeOH (44:56) at constant flow and pressure. Van't Hoff plots and thermodynamic quantities calculated from them indicate that significant conformational changes in insulin molecule in TFA-ACN begin at ~ 53 °C, while these are already present at lower temperatures in TFA-MeOH. Similarly, significant conformational changes of cytochrome c in TFA-ACN are present at lower temperatures, but do not occur in TFA-MeOH. Lysozyme and BSA were analysed with gradient method 0.1 % TFA-ACN. Trends in retention times indicate that conformational changes occur in these two proteins as well.

• Keywords: protein chromatography, non-linear van't Hoff plot, insulin, cytochrome, lysozyme, BSA