Binding of iron by factor IX. Possible role for beta-hydroxyaspartic acid

Area of Science:

• Biochemistry
• Hematology
• Protein Chemistry

Background:

• Bovine factor IX is a key protein in the blood coagulation cascade.
• The presence of metal ions can influence protein structure and function.
• Understanding metal binding in coagulation factors is important for hematology research.

Purpose of the Study:

• To investigate the presence and nature of metal binding in bovine factor IX.
• To determine the affinity and specificity of iron binding to factor IX.
• To identify potential metal-chelating residues within factor IX.

Main Methods:

• UV-Vis spectroscopy to detect color and absorption maxima.
• X-ray fluorescence to identify elemental composition.
• Atomic absorption spectroscopy for quantitative metal analysis.
• EDTA chelation assay to assess iron dissociation kinetics.

Main Results:

• Bovine factor IX exhibits a pink color at high concentrations due to iron binding (λmax = 500 nm).
• Iron is the only transition metal detected in factor IX preparations.
• Factor IX binds iron with a 1:1 molar ratio (1 g atom Fe/mol factor IX).
• Iron is tightly bound, with slow dissociation by EDTA (t1/2 = 3 h).
• Prothrombin shows no detectable iron binding, and factor X binds <0.2 g atom Fe/mol.

Conclusions:

• Bovine factor IX possesses a high-affinity iron-binding site.
• The beta-hydroxyaspartate residue is proposed as the iron-chelating moiety.
• Iron binding to factor IX may have physiological significance in hemostasis.