Vplivi pufrov in soli na lastnosti proteinov BSA in HEWL

Jaklin, Matej (author), Hribar Lee, Barbara (mentor)

Proteini so sestavljeni iz dolgih verig aminokislin. Njihova struktura in stabilnost sta potrebni za obstoj živih organizmov. Aktivni so le v svoji nativni konformaciji. Na konformacijo, strukturo ter stabilnost raztopin proteinov lahko vpliva mnogo parametrov. Med glavne spadajo pH, temperatura, vrsta in koncentracija pufra ter koncentracija specifičnih dodanih snovi (soli, sladkorji, denaturanti…). Vodne raztopine proteinov običajno vsebujejo pufre, ki uravnavajo pH raztopine in zagotavljajo stabilnost proteinov. Poleg pufrov raztopine običajno vsebujejo tudi nekatere nizko molekularne soli. Pufer na protein ne vpliva le z uravnavanjem pH raztopine, temveč se nanj lahko veže in spremeni njegove lastnosti. Vezava je bila opažena tudi pri preučevanju vpliva ionov soli na protein. Namen magistrske naloge je povečati razumevanje interakcij protein-protein, protein-ion ter protein-pufer. Preverjal sem, kako sprememba različnih parametrov (koncentracije in vrste pufra, koncentracije proteina ter pH) vpliva na zeta potencial in velikosti delcev raztopine proteina HEWL. Rezultate meritev velikosti delcev in zeta potenciala sem povezal z vezavo molekul pufra na površino proteina. Pri pH 2 sem kot pufer uporabljal fosfat, glicin ter KCl-HCl. Pri pH 9 pa sem za pufra uporabil glicin in bis tris. Koncentracije pufrov sem študiral v območju koncentracij od 0,01 M do 0,5 M. Vpliv koncentracije proteina sem spremljal od 0,5 mg/ml do 2,5 mg/ml. Vse ostale raztopine so imele koncentracijo proteina 5 mg/ml. Na podoben način sem raziskoval tudi vplive na protein BSA. V primeru BSA sem se posvečal raziskovanju vpliva dodatka različnih vrst in koncentracij (0,1 M do 1,75 M) alkalijskih soli (NaCl, NaBr, NaI, LiCl, KCl, RbCl ter CsCl). Preučeval sem skupni vpliv vrste pufra in vrste soli na velikost delcev in zeta potenciala. Pri pH 4,3 sem izvedel dve seriji eksperimentov, pri čemer sem enkrat uporabil 20 mM acetatni pufer, drugič pa citratnega. Rezultate teoretično izračunanih vezavnih energij (ki sem jih izračunal s pomočjo analize molekulskega sidranja oz. »dockinga«) sem povezal z izmerjenimi vrednostmi velikosti delcev in zeta potenciala. Sidranje sem izvedel le na proteinu HEWL, ki je v primerjavi z BSA manj kompleksen.

• Language: Slovenian
• Keywords: Protein, pufer, soli, BSA, HEWL
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2020
• PID: 20.500.12556/RUL-120281
• COBISS.SI-ID: 33685251
• Publication date in RUL: 17.09.2020

Secondary language

• Language: English
• Title: The influence of salts and buffers on the properties of proteins BSA and HEWL

Abstract

Proteins are made up of long chains of amino acids. Their structure and stability are important for the existence of life. They are only active in their native conformation, which together with their structure and stability depends on a large number of parameters. These include above all pH value, temperature, type and concentration of the buffer and the concentration of specific added substances (salt, sugar, denaturants ...). Aqueous solutions of proteins usually contain buffers that regulate the pH of a solution and maintain the stability of the protein. Solutions usually contain some low molecular weight salts. The buffer not only influences the protein by regulating the pH, but can also bind to the protein and change its properties. Similar binding has also been observed when studying the effects of ions (from used salts) on the protein. The aim of this work is to deepen the understanding of protein-protein, protein-ion and protein-buffer interactions. We investigated how the change of different parameters (concentration and type of buffer, concentration of the protein and pH) affects the zeta potential and the size of the particles in the HEWL protein solution. We correlated the measurements with the binding of buffer molecules on the surface of the protein. At pH 2 we used phosphate, glycine and KCl-HCl as buffers. At pH level 9 we used glycine and bis tris. We also investigated the effect of changing the protein concentration from 0.5 mg/ml to 2.5 mg/l. All other solutions had a protein concentration of 5 mg/ml. In a similar way we investigated the effects on the BSA protein. In the case of BSA, we devoted our efforts to studying the effects of adding different types and concentrations (0.1 M - 1.75 M) of alkali salts (NaCl, NaBr, NaI, LiCl, KCl, RbCl and CsCl). We investigated the overall influence of the type of buffer and the type of salt on particle size and zeta potential. At pH 4.3 we performed two sets of experiments, one with 20mM acetate buffer and the other with citrate. The results of the theoretically calculated binding energies (calculated by molecular docking analysis) were related to the measured particle sizes and zeta potentials. Only the HEWL protein was analysed by docking analysis because the BSA structure is too complex.

• Keywords: Proteins, buffers, salts, BSA, HEWL