High-performance liquid affinity chromatography on silica-bound alcohol dehydrogenase

Area of Science:

• Biochemistry
• Chromatography
• Enzyme immobilization

Background:

• Enzyme immobilization is crucial for developing reusable biocatalysts.
• Affinity chromatography requires stable and active immobilized enzymes.
• Horse liver alcohol dehydrogenase (HLADH) is a well-characterized enzyme.

Purpose of the Study:

• To immobilize HLADH onto tresyl chloride-activated glycerylpropyl-silica.
• To characterize the immobilized enzyme's properties and binding sites.
• To evaluate its performance in High-Performance Liquid Chromatography (HPLC).

Main Methods:

• Immobilization of HLADH on 10-micron, 1000-A pore glycerylpropyl-silica using tresyl chloride activation.
• Scatchard plot analysis and active-site titrations for enzyme characterization.
• HPLC separation of adenine nucleosides, adenine nucleotides, and triazine dyes.

Main Results:

• Achieved near 100% coupling and activity yield after immobilization.
• Coenzyme-binding sites remained largely unaffected, showing equivalent accessibility.
• Successful chromatographic separation of target molecules with calculated dissociation constants matching literature values.
• Demonstrated predictable effects of nucleotide derivatives on binding strength.

Conclusions:

• Tresyl chloride-activated silica is an effective support for HLADH immobilization.
• The immobilized enzyme retains significant activity and binding site integrity.
• Silica-bound HLADH is suitable for affinity chromatography applications in HPLC.