Multiple forms of carbonic anhydrase in Tetragonia tetragonioides leaves and the impact of heavy metal ions on enzyme activity.

Tetragonia tetragonioides (Aizoaceae; Caryophyllales), an annual herbaceous plant, a halophyte native to the marine coasts of New Zealand is now cultivated worldwide as a minor salt-tolerant crop. The aim of this study was to evaluate the activity of carbonic anhydrase (CA, EC 4.2.1.1) and identify the CA isoforms in a fraction of total soluble proteins from T. tetragonioides leaves and to determine CA sensitivity to specific sulfonamide inhibitors and heavy metal (HM) ions. In higher plants, CA is one of the most abundant leaf enzymes catalyzing CO and HCO interconversions that maintain the balance between dissolved forms of inorganic carbon. Catalytically active CA isoforms in the soluble protein fraction isolated from T. tetragonioides leaves were identified by a protonography method which involves the detection of CA activity in the gel following SDS-PAGE and subsequent removal of SDS (De Luca et al., 2015). This approach enabled the detection of active oligomeric forms of CA in the gel, allowing for the separate assessment of their activity. The protonogram revealed the presence of five sources of CA activity, which correspond to isoforms with approximate molecular masses of 26, 35, 41, 52, and 166 kDa. The total CA activity of T. tetragonioides leaf proteins was found to be inhibited by specific CA inhibitors, acetazolamide (AZ) and ethoxyzolamide (EZ), within the same concentration range as CAs from other plants. The half-maximal inhibition of hydratase activity was estimated to be 16 μM AZ and 2 μM EZ. The in vitro impact of selected heavy metal ions (Cu⁺, Cd⁺, Zn⁺, Pb⁺, Hg⁺, and Ag⁺) on the hydratase activity of the soluble T. tetragonioides protein fraction was examined. High sensitivity of CA activity to inhibition by silver (I50-0.5 μM) and mercury ions (I ∼ 2 μM) was shown. Copper and cadmium ions inhibited CA activity with I50 ∼ 40 and ∼12 μM, respectively. Zinc ions (I50 ∼ 200 μM) were weaker inhibitors, and lead ions in the concentration range of 50-500 μM insignificantly stimulated the hydratase activity of soluble proteins of T. tetragonioides. The high sensitivity of T. tetragonioides CA to Ag⁺ and Hg⁺ ions, which are known to act as sulfhydryl poisons, can be attributed to the presence of SH-containing cysteines within the active site of the plant CAs.