The fraction of stromal proteins was obtained after osmotic destruction of whole chloroplasts isolated from spinach leaves (Spinacia oleracea) and washed from the cytosol components. The effect of cadmium ions on the activity of stromal carbonic anhydrase (CA) was studied by infrared CO2 analysis. The half-maximal inhibition of CA dehydratase activity was observed under 35 µM CdCl2 in the solution; at 80 µM CdCl2, the enzyme activity was 15 % of the control. The CA activity in PAAG after non-denaturing electrophoresis was visualized by the change in the color of the bromothymol blue indicator at the sites of CA localization. Four protein zones with different mobility and different levels of CA activity were identified. The number of zones with CA activity and the intensity of their staining decreased after pre-incubation of the stromal proteins with CdCl2, depending on its concentration. The low molecular weight form of CA was the most sensitive to the Cd2+ action, whose activity was completely suppressed in the samples treated with 80 µM CdCl2, while the CA migrated together with Rubisco remained partly active after stromal proteins incubation with 150 µM CdCl2. The obtained data confirm the possibility of chloroplast CA using as a biomarker for early monitoring of the environmental pollution by heavy metals.
Keywords: Spinacia oleracea, cadmium, carbonic anhydrase, biomarker
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