Interaction of native and apo-carbonic anhydrase with hydrophobic adsorbents: A comparative structure-function study

Area of Science:

• Biochemistry
• Enzymology
• Protein Chemistry

Background:

• Native carbonic anhydrase exhibits limited interaction with hydrophobic adsorbents.
• Denaturation of carbonic anhydrase enhances its interaction with hydrophobic surfaces.

Purpose of the Study:

• To investigate the characteristic features of apo carbonic anhydrase compared to its native form.
• To assess the binding affinity of apo carbonic anhydrase to hydrophobic adsorbents.
• To develop catalytically active immobilized carbonic anhydrase preparations.

Main Methods:

• Preparation of apo carbonic anhydrase by zinc removal.
• Characterization using far- and near-UV circular dichroism (CD).
• Intrinsic fluorescence spectroscopy, 1-anilino naphthalene-8-sulfonate (ANS) binding, fluorescence quenching, and Tm measurement.

Main Results:

• Apo carbonic anhydrase exhibits increased protein flexibility and more exposed hydrophobic sites.
• The apo form shows a significantly greater affinity for hydrophobic adsorbents than the native form.
• Heat denaturation of the apo form facilitates interaction with adsorbent alkyl residues, enabling zinc re-addition for active immobilization.

Conclusions:

• Apo carbonic anhydrase possesses enhanced properties for hydrophobic interactions.
• Immobilization strategies can be optimized by utilizing the apo form and controlled denaturation/renaturation.
• This study provides a pathway for creating functional immobilized enzyme systems.