Catalytically active azoaldolase. Preparation on solid support

Area of Science:

• Biochemistry
• Enzyme engineering

Background:

• Rabbit muscle aldolase is a key glycolytic enzyme.
• Enzyme modification can alter protein properties and functions.

Purpose of the Study:

• To describe the coupling of p-carboxy benzene diazonium chloride with rabbit muscle aldolase.
• To characterize the spectroscopic, structural, and catalytic features of the resulting azoenzyme.

Main Methods:

• Coupling reaction at pH 7.2.
• Support on phosphocellulose.
• Disc gel electrophoresis (including with 8 M urea).
• Spectroscopic studies and amino acid analysis.

Main Results:

• A homogeneous tetrameric azoenzyme was successfully prepared.
• Twelve molecules of the reactant were bound per protein molecule.
• Eight azocysteine and one azohistidine residues were identified.
• The azoaldolase maintained catalytic activity with its natural substrates.
• The enzyme's pH activity profile broadened slightly in the alkaline region without affecting physiological pH activity.

Conclusions:

• The described procedure yields a stable and homogeneous azoenzyme.
• Enzyme modification did not compromise essential catalytic functions.
• The modified enzyme exhibits potential for further biochemical studies and applications.