Measuring carbonic anhydrase activity in alpha-carboxysomes using stopped-flow

Area of Science:

• Biochemistry
• Molecular Biology
• Microbiology

Background:

• Carboxysomes are essential protein organelles in bacteria for carbon dioxide concentration.
• They house carbonic anhydrase and Rubisco, crucial for efficient CO2 fixation.
• The carbon dioxide concentration mechanism (CCM) enhances Rubisco's carboxylation activity.

Purpose of the Study:

• To present a general method for purifying alpha-carboxysomes.
• To describe a technique for measuring carbonic anhydrase activity within purified carboxysomes.
• To determine kinetic parameters of carbonic anhydrase in alpha-carboxysomes.

Main Methods:

• Purification of alpha-carboxysomes from Halothiobacillus neapolitanus c2.
• Utilizing the Khalifah/pH indicator assay for carbonic anhydrase activity measurement.
• Employing a stopped-flow spectrometer for rapid kinetic analysis of CO2 hydration.

Main Results:

• Successfully purified alpha-carboxysomes and measured their carbonic anhydrase activity.
• Determined key kinetic parameters (kcat, KM, kcat/KM) for carbonic anhydrase.
• Demonstrated the applicability of the method for various carboxysome sources.

Conclusions:

• The presented method provides a robust approach for studying carboxysome-associated carbonic anhydrase.
• This technique facilitates deeper understanding of the bacterial CCM.
• The protocol is adaptable for diverse carboxysome research and carbonic anhydrase studies.