Usporedba metoda za mjerenje koncentracije proteina u realnim uzorcima

undergraduate thesis

• Title (english): Comparison of methods for measurement of protein concentration in real samples
• Author: Marija Berečić
• Mentor: Anita Šalić (mentor)
• Mentor: Zvjezdana Findrik Blažević (komentor)
• Committee member: Anita Šalić (predsjednik povjerenstva)
• Committee member: Zvjezdana Findrik Blažević (član povjerenstva)
• Committee member: Domagoj Vrsaljko (član povjerenstva)
• Granter: University of Zagreb; Faculty of Chemical Engineering and Technology; Zagreb
• Defense date and country: 2022-09-23, Croatia
• Scientific / art field, discipline and subdiscipline: TECHNICAL SCIENCES; Chemical Engineering

Abstract

Proteini su biološke makromolekule koje su izgrađene od aminokiselina povezanih peptidnim vezama. Osim što su ključne komponente metabolizma živih organizama, mnogi se proteini mogu koristiti u terapeutske ili dijagnostičke svrhe, ali i u mnogim drugim područjima kao što su kozmetička i prehrambena industrija. Kako bi njihova upotreba bila sigurna, bitno je točno odrediti njihove koncentracije. Danas se u praksi koriste različite metode određivanja koncentracije proteina, međutim, najčešće korištene su Biuret metoda, Lorwryeva metoda i Bradfordova metoda jer su brze, jednostavne i jeftine. Glavni nedostatak spomenutih metoda je to što na točnost mjerenja mogu utjecati druge molekule koje su prisutne u otopini proteina. Cilj ovoga rada je ispitati i usporediti osjetljivost Biuret metode, Lorwryeve metode i Bradfordove metode za određivanje niskih koncentracija proteina te osjetljivost pojedine metode na prisutnost različitih molekula u otopini proteina. U ovom radu istražen je utjecaj soli, metala, šećera, deterdženata, otapala, kiselina i baza te drugih tvari (DMSO, EDTA) na točnost mjerenja Biuret metode, Lowryjeve metode i Bradfordove metode. Pripremljene su dvije različite koncentracije modelne otopine goveđeg serumskog albumina (BSA) (γBSA = 5 i 50 mg/L), na kojima su provedena sva ispitivanja. Dobiveni rezultati pokazali su kako velik broj istraženih komponenti smanjuje točnost odabranih metoda. Na samom je kraju određena koncentracija proteina u sirovom ekstraktu enzima lipaze i ksilanaze (bez dodatnih stupnjeva pročišćavanja) proizvedenih fermentacijom Thermomyces lanuginosus na pogači buče.

Abstract (english)

Proteins are biological macromolecules composed of amino acids linked by peptide bonds. Not only are they the main metabolic products of living organisms, but many of them can be used for therapeutic or diagnostic purposes and in many other areas such as the cosmetics and food industries. In order to use them safely, it is important to determine their concentrations accurately. In current practice, various methods are used to determine their concentrations. However, the most commonly used are the Biuret assay, the Lowry assay, and the Bradford assay, as they are rapid, easy to use, and inexpensive. The major drawback of the aforementioned assays is that the accuracy of the measurement can be affected by other molecules present in the protein solution. The aim of this study is to test and compare the sensitivity of the Biuret assay, the Lowry assay and the Bradford assay for the determination of a low protein concentration and the sensitivity of each assay to the presence of different molecules in the protein solution. This study investigates the influence of some compounds (salts, metals, sugars, detergents, organic solutions, acids and bases, DMSO and EDTA) on the accuracy of the Biuret assay, the Lowry assay and the Bradford assay. Two different concentrations of a standard solution of bovine serum albumin (BSA) (γBSA = 5 i 50 mg/L ) were prepared, with which all assays were performed. The results obtained showed that a large number of compounds tested reduced the accuracy of the chosen methods. At the very end, the protein concentration in the crude extract of lipase and xylanase enzymes (without additional purification steps) produced by fermentation of Thermomyces lanuginosus on pumpkin cake was determined.

• Keywords: protein; određivanje koncentracije; Biuret metoda; Lorwryeva metoda; Bradfordova metoda
• Keywords (english): protein; determination of concentration; Biuret assay; Lowry assay; Bradford assay
• Language: croatian
• URN:NBN: urn:nbn:hr:149:671496
• Study programme: Title: Environmental Engineering - Undergraduate study; Study programme type: university; Study level: undergraduate; Academic / professional title: sveučilišni/a prvostupnik/ prvostupnica (baccalaureus/baccalaurea) inženjer/inženjerka ekoinženjerstva (sveučilišni/a prvostupnik/ prvostupnica (baccalaureus/baccalaurea) inženjer/inženjerka ekoinženjerstva)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2023-02-17 13:39:39