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Microplate assay for endo-polygalacturonase activity determination based on ruthenium red method
Gastón E Ortiz1, María E Guitart2, Edgardo Albertó1
1Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico Chascomús (IIB-INTECH, UNSAM-CONICET), Universidad de San Martín, San Martín, Buenos Aires 1650, Argentina.
Analytical Biochemistry
|March 12, 2014
Summary
This study introduces a high-throughput assay for endo-polygalacturonase (endo-PGase) activity using ruthenium red. The new method offers a faster alternative to traditional viscosity measurements for enzyme determination.
Area of Science:
- Enzymology
- Biochemistry
- Analytical Chemistry
Background:
- Enzyme activity assays for endo-polygalacturonase (endo-PGase) traditionally rely on viscosity changes or quantifying reducing sugars.
- Ruthenium red (RR) has been identified as a useful reagent for endo-PGase determination.
Purpose of the Study:
- To develop and validate a high-throughput, liquid-based assay for endo-PGase activity.
- To compare the performance of the new RR-based assay with the conventional viscosity determination method.
Main Methods:
- A high-throughput liquid-based assay utilizing ruthenium red (RR) was developed.
- The assay employs small volumes of enzyme solution, polygalacturonic acid, and RR.
- Assay performance was compared against the standard viscosity determination method.
Main Results:
- A high-throughput liquid-based assay for endo-PGase was successfully established.
- The RR-based assay demonstrated comparable results to the viscosity method.
- An interconversion factor between RR and viscosity activities was determined.
Conclusions:
- The ruthenium red-based assay provides a viable, high-throughput alternative for quantifying endo-PGase activity.
- This method simplifies enzyme activity determination and facilitates higher sample throughput.
- The established interconversion factor allows for direct comparison between the two assay methodologies.
