Borate-nucleotide complex formation depends on charge and phosphorylation state.

Area of Science:

• Analytical Chemistry
• Biochemistry
• Mass Spectrometry

Background:

• Borate complexation with biomolecules is crucial for understanding molecular interactions.
• Nucleotide structure, including charge and phosphorylation, influences binding affinities.
• Electrospray ionization mass spectrometry (ESI-MS) is a powerful tool for studying non-covalent interactions.

Purpose of the Study:

• To investigate the formation and characteristics of borate-nucleotide complexes.
• To determine how nucleotide structure affects borate binding affinity.
• To assess the physiological relevance of observed borate-nucleotide complexes.

Main Methods:

• Flow injection analysis coupled with electrospray ionization mass spectrometry (FIA-ESI-MS).
• Preparation of solutions with varying nucleotide types and concentrations in a water-acetonitrile-triethylamine solvent system.
• Determination of association constants (K(A)) for borate complexes with adenine, guanine, cytidine, and uridine nucleotides at different pH values.

Main Results:

• Borate complexation with nicotinamide nucleotides (NAD+, NADH, NADP+, NADPH) is influenced by the charge on the nicotinamide group and the number of phosphate groups.
• Borate binding affinity decreases with increasing numbers of phosphate groups in adenine nucleotides (AMP, ADP, ATP) and other nucleoside phosphates.
• At physiological pH (7.4), only the borate-NAD+ complex was detected, suggesting its higher stability and relevance.

Conclusions:

• Nucleotide phosphorylation level is inversely proportional to borate complex abundance.
• The borate-NAD+ complex is the most significant and physiologically relevant among the studied borate-nucleotide interactions.
• FIA-ESI-MS provides valuable insights into the binding dynamics of borate with various nucleotides.