Rapid, direct, noninvasive method to determine the amount of immobilized protein

Ambrožič, Rok (author), Mravljak, Rok (author), Podgornik, Aleš (author)

Protein immobilization is of utmost importance in many areas, where various proteins are used for selective detection of target compounds. Despite the importance given to determine the amount of immobilized protein, there is no simple method that allows direct, noninvasive detection. In this work, a method based on pH transition, occurring during change of solution ionic strength, was developed. The method utilized the ionic character of the immobilized protein while implementing biologically compatible buffers. Five different proteins, namely, glucose oxidase, horseradish peroxidase, bovine serum albumin, lysozyme, and protein A, were immobilized in different amounts on a porous polymeric matrix, and their pH transition was measured using lactate buffer of various concentrations and pH values. A linear correlation was found between the amount of immobilized protein and the amplitude of the pH transition, allowing the detection down to 2 nmol of immobilized protein. By changing the buffer concentration and pH, the sensitivity of the method could be tailored. Criteria based on the symmetry of the pH transition peak have been developed to determine if a particular measurement is within a linear range. In addition, a mathematical model was developed enabling prediction of pH transition profiles based solely on the protein amino acid sequence, the buffer pK$_a$ value(s), and the amount of immobilized protein. Hence, it can be used to design pH transition method experiments to achieve the required sensitivity for a target sample. Since the proposed method is noninvasive, it can be routinely applied during optimization of the immobilization protocol, for quality control, and also as an in-process monitoring tool.

• Language: English
• Keywords: immobilization, pH transition, protein quantity, monomers, peptides, proteins, theoretical chemistry, computational chemistry, pH
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Publication status: Published
• Publication version: Version of Record
• Year: 2023
• Number of pages: Str. 5643-5651
• Numbering: Vol. 95, iss. 13
• PID: 20.500.12556/RUL-146106
• UDC: 66.098
• ISSN on article: 0003-2700
• DOI: 10.1021/acs.analchem.2c05402
• COBISS.SI-ID: 147263491
• Publication date in RUL: 19.05.2023

Record is a part of a journal

• Title: Analytical chemistry
• Shortened title: Anal. chem.
• Publisher: American Chemical Society
• ISSN: 0003-2700
• COBISS.SI-ID: 5085703

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: imobilizacija, pH prehod, količina proteina