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Rubisco Extraction and Purification from Diatoms.

Jodi N Young1, Ana M C Heureux2, Rosalind E M Rickaby2

  • 1Department of Oceanography, University of Washington, Seattle, USA.

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|August 30, 2021
PubMed
Summary

This study details a method for extracting ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) from diatoms. The protocol allows for measuring Rubisco

Keywords:
Carbon fixationDiatomsExtractionPhytoplanktonRubisco

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Area of Science:

  • Biochemistry
  • Marine Biology
  • Plant Physiology

Background:

  • Ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) is a key enzyme in carbon fixation.
  • Diatoms (Bacillophyta) are significant primary producers in marine ecosystems.
  • Accurate measurement of Rubisco's catalytic performance is crucial for understanding diatom productivity.

Purpose of the Study:

  • To describe a standardized protocol for extracting Rubisco from diatoms.
  • To enable the determination of Rubisco's catalytic parameters, including carboxylation and oxygenation kinetics.
  • To facilitate measurements of Rubisco's CO2/O2 specificity in diatoms.

Main Methods:

  • Adaptation of established Rubisco extraction protocols from cyanobacteria and higher plants.
  • A two-part extraction process: initial crude extract for basic assays and further purification for specificity measurements.
  • Carboxylation assays to determine Michaelis constant for CO2 (KC) and catalytic turnover rate (k).

Main Results:

  • A reproducible method for obtaining crude diatom Rubisco extracts.
  • The crude extract is sufficient for determining KC and k values.
  • Additional purification steps enable the measurement of Rubisco's CO2/O2 Specificity (SC/O).

Conclusions:

  • The described protocol provides a reliable method for Rubisco extraction and characterization in diatoms.
  • This method supports detailed enzymatic analysis of Rubisco from Bacillophyta.
  • Facilitates comparative studies on Rubisco function across different photosynthetic organisms.