Hemoglobin Removal by Zinc Sulfate to Assay Level and Activity of Red Blood Cell Caspase 3 Using ELISA and Colorimetric Methods

Zinc sulfate binds to the R group of some amino acids, such as histidine and cysteine, resulting in protein precipitation. In an ELISA and colorimetric experiments, we determined the optimal concentration of zinc sulfate to precipitate hemoglobin, which in turn affects the level and activity of Caspase 3 in red blood cells.

Osmotic stress was induced on red blood cells under hypertonic and hypotonic conditions. An isotonic condition was used as a control without osmotic stress. The cells were incubated at 37oC for 15 min and 24 hrs. Different concentrations of zinc sulfate were set up experimentally, stepwise after the lysis of RBC samples with ultrasound waves and removal of cell membranes by centrifugation. Zinc sulfate was allowed to bind to hemoglobin at different time intervals at room temperature. Afterward, hemoglobin was precipitated at various time intervals through centrifugation. The supernatants were then measured by ELISA and colorimetric methods for Caspase 3 level and activity.

The optimal conditions were found to be 6 mM zinc sulfate, 10 min incubation at room temperature to bind zinc sulfate to hemoglobin, and 30 min centrifugation at 3000 rpm to precipitate hemoglobin.

This study showed that zinc sulfate with a concentration of 6 mM precipitates and removes hemoglobin without affecting the level or activity of Caspase 3.