New method for guanase activity measurement by high-performance liquid chromatography

Area of Science:

• Biochemistry
• Analytical Chemistry

Background:

• Guanase (EC 3.5.4.3) is an enzyme involved in purine metabolism.
• Accurate and rapid determination of enzyme activity is crucial for biochemical research and diagnostics.

Purpose of the Study:

• To develop and validate a rapid, reproducible high-performance liquid chromatographic (HPLC) method for determining guanase activity.
• To enable sensitive detection of substrate and product concentrations for kinetic analysis.

Main Methods:

• Isocratic high-performance liquid chromatography (HPLC) was employed for the separation and quantification of guanase reaction components.
• The method was optimized for speed and reproducibility, requiring approximately 10 minutes for complete analysis.
• Detection limits allowed for nanomolar concentration changes of substrate and product.

Main Results:

• The developed HPLC method demonstrated high reproducibility, with a coefficient of variation below 1%.
• Kinetic studies yielded apparent Michaelis constants (Km) of 13.3 µM (Tris-HCl buffer) and 8.5 µM (phosphate buffer) at 37°C.
• Maximum reaction rates (Vmax) were determined as 1.95 pmol/min/mg protein in Tris-HCl and 3.84 pmol/min/mg protein in phosphate buffer.

Conclusions:

• The proposed HPLC method provides a rapid, accurate, and sensitive tool for guanase activity determination.
• This method facilitates detailed kinetic studies of guanase under different buffer conditions.
• The technique's efficiency and sensitivity make it suitable for various biochemical and diagnostic applications.