One-minute high-performance liquid chromatography assay for 5'-nucleotidase using a 20-mm reverse-phase column

Area of Science:

• Biochemistry
• Analytical Chemistry
• Enzymology

Background:

• Nucleotidases are crucial enzymes involved in nucleotide metabolism.
• Existing assays for nucleotidase activity often lack sensitivity or require radioactive materials.
• Accurate determination of nucleotidase activity is important for understanding various physiological and pathological processes.

Purpose of the Study:

• To develop and validate a novel, sensitive, and non-radioactive High-Performance Liquid Chromatography (HPLC)-based assay for quantifying nucleotidase activity.
• To apply this method for the determination of pyrimidine 5 andomIndex-nucleotidase activity in human erythrocyte lysate and plasma.

Main Methods:

• Utilized a C-18 reversed-phase column with isocratic elution for rapid separation of substrate and product.
• Employed High-Performance Liquid Chromatography (HPLC) with ultraviolet (UV) detection for quantitation.
• Optimized mobile phase conditions using 100 mM potassium phosphate (pH 6.0) at a flow rate of 2 ml/min.

Main Results:

• Achieved separation of nucleoside products from nucleotide substrates in under 1 minute.
• Demonstrated high sensitivity, being 50-fold more sensitive than colorimetric phosphate measurements.
• Showed excellent agreement with results obtained by measuring inorganic phosphate production.
• The assay does not necessitate prior sample dialysis.

Conclusions:

• The developed HPLC assay provides a rapid, sensitive, and non-radioactive method for determining nucleotidase activity.
• This versatile method is suitable for various biological sources and offers significant advantages over traditional assays.
• The assay is particularly effective for pyrimidine 5 andomIndex-nucleotidase activity assessment in human erythrocytes and plasma.