| Summary: | Carbonic anhydrase (CA) plays an important physiological role in all biological systems by accelerating the interconversion of CO2 and HCO3−. In algae, CA is essential for photosynthesis: external CA (CAext) dehydrates HCO3−, enhancing the supply of CO2 to the cell surface, and internal CA (CAint) interconverts HCO3− and CO2 to maintain the inorganic carbon (Ci) pool and supply CO2 to RuBisCO. We first conducted a literature review comparing the conditions in which CA extraction and measurement have been carried out, using the commonly used Wilbur–Anderson method. We found that the assay has been widely modified since its introduction in 1948, mostly without being optimized for the species tested. Based on the review, an optimized protocol for measuring CA in Macrocystis pyrifera was developed, which showed that the assay conditions can strongly affect CA activity. Tris–HCl buffer gave the highest levels of CA activity, but phosphate buffer reduced activity significantly. Buffers containing polyvinylpyrrolidone (PVP) and dithiothreitol (DTT) stabilized CA. Using the optimized assay, CAext and CAint activities were readily measured in Macrocystis with higher precision compared to the non-optimized method. The CAint activity was 2 × higher than CAext, which is attributed to the Ci uptake mechanisms of Macrocystis. This study suggests that the CA assay needs to be optimized for each species prior to experimental work to obtain both accurate and precise results.
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