Improved method for estimation of inorganic phosphate: implications for its application in enzyme assays.

Area of Science:

• Biochemistry
• Analytical Chemistry

Background:

• The conventional Fiske and Subba Row method for inorganic phosphate (Pi) estimation provides rapid results but suffers from unstable color, limiting sample throughput.
• Existing modifications have failed to resolve the color stability issue and can be unsuitable for enzyme assays using acid-labile phosphate substrates due to high sulfuric acid concentrations.

Purpose of the Study:

• To modify the Fiske and Subba Row method for enhanced color stability and sensitivity.
• To ensure the method's applicability in enzyme assays, particularly those involving acid-labile phosphate-containing substrates like ATP.

Main Methods:

• Utilized higher concentrations of sulfuric acid (0.625, 0.8, and 1.0 N) compared to the conventional 0.5 N.
• Evaluated color development, stability, and absorption maxima over 24 hours.
• Assessed the impact of common assay components (sugars, salts, MgCl2, BSA) and substrate hydrolysis (ATP, G6P) on the modified method.

Main Results:

• The modified method demonstrated stable color development for up to 24 hours, with optimal intensity achieved at 24 hours.
• Absorption maximum shifted from 660 nm to 820 nm, and sensitivity increased by 1.7-2.1 times at 15 minutes and 4.8-fold at 24 hours.
• The assay showed no interference from common buffer components and minimal hydrolysis of ATP and negligible hydrolysis of G6P.

Conclusions:

• The modified Fiske and Subba Row method with increased sulfuric acid concentration offers superior color stability and sensitivity.
• This enhanced method is suitable for inorganic phosphate analysis in enzyme assays, even with acid-labile phosphate substrates like ATP.