Determination, purification, and characterization of alpha-NADH

Area of Science:

• Biochemistry
• Analytical Chemistry

Background:

• Alpha-NADH and beta-NADH are crucial coenzymes with distinct roles.
• Accurate quantification and purification of alpha-NADH are essential for biochemical research.
• Existing methods for alpha-NADH analysis and preparation have limitations.

Purpose of the Study:

• To develop a reliable method for alpha-NADH determination in complex mixtures.
• To establish a high-purity alpha-NADH preparation protocol.
• To investigate the kinetics of alpha- and beta-NADH anomerization.
• To characterize the spectral properties of purified alpha-NADH.

Main Methods:

• Anion exchange High-Performance Liquid Chromatography (HPLC) combined with enzymatic oxidation.
• Preparative anion exchange chromatography coupled with reversed-phase HPLC for purification.
• Kinetic studies at varying pH to determine anomerization rate constants.
• Spectrophotometric and fluorometric analysis at different temperatures.

Main Results:

• A novel HPLC method accurately quantifies alpha-NADH even with high beta-NADH concentrations.
• Purification yields alpha-NADH with 95-98% purity, surpassing commercial preparations (80-90%).
• Anomerization rate constants (kappa 1 and kappa 2) and equilibrium constants were determined at pH 7.0 and 7.3.
• Precise absorption and fluorescence maxima for alpha-NADH were newly established.

Conclusions:

• The developed HPLC method is suitable for impurity detection and accurate alpha-NADH quantification.
• The purification strategy provides a reliable source of high-purity alpha-NADH.
• Understanding anomerization kinetics is vital for interpreting NADH-dependent enzymatic reactions.
• Newly determined spectral data enhance the characterization of alpha-NADH.