A novel simple fluorometric protease assay for monitoring hydrolysis of proteins in real time

Bahun, Miha (author), Poklar Ulrih, Nataša (author)

Measuring the activity of proteases is essential for investigating both the physiological functions and commercial applications of these enzymes. In contrast to the numerous protease assays that are based on chromogenic or fluorogenic peptide substrates, there is a lack of approaches to monitor degradation of proteins in real time. Here we report a protease assay where SYPRO Orange is employed as a fluorogenic probe to follow proteolysis. The functionality of the assay was demonstrated with the two subtilases of varying thermostability, using four different protein substrates. The assay is compatible with a real-time PCR instrument which allows continuous fluorescence measurements in low-volume samples even at high temperatures. This makes the assay especially suitable for high-throughput characterization of thermostable proteases.

• Language: English
• Keywords: protease assay, proteolysis, subtilisin, SYPRO orange, fluorescence, real-time PCR, thermostability
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: BF - Biotechnical Faculty
• Publication status: Published
• Publication version: Version of Record
• Year: 2025
• Number of pages: 9 str.
• Numbering: Vol. 696, art. 115688
• PID: 20.500.12556/RUL-164279
• UDC: 577.152.34
• ISSN on article: 0003-2697
• DOI: 10.1016/j.ab.2024.115688
• COBISS.SI-ID: 212076803
• Publication date in RUL: 18.10.2024

Record is a part of a journal

• Title: Analytical biochemistry
• Shortened title: Anal. biochem.
• Publisher: Elsevier
• ISSN: 0003-2697
• COBISS.SI-ID: 6506759

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: encimi, proteaze, aktivnost proteaz, proteoliza, PCR

Projects

• Funder: ARIS - Slovenian Research and Innovation Agency
• Project number: P4-0121
• Name: Biokemijska in biofizikalno-kemijska karakterizacija naravnih snovi