**2.2 Preparation of crude purified hemoglobin**

For purified hemoglobin preparation, pRBCs tube was placed in a dry-ice acetone bath for few seconds until the PRBCs freeze and visibly seen as solid. The tube immediately was removed from the dry-ice acetone bath to thaw in the hot water bath. Sudden cooling and thawing three times cause proteins of erythrocyte's membrane to denature and eventually lead to cell lysis. After the pRBCs are lysed, they will be placed in the PD 10 desalting column. The PD-10 desalting columns are intended for the fast removal of proteins and other big macromolecules from samples [3]. The PD-10 desalting columns are used to capture large-molecular weight chemicals and proteins, particularly glutathione (307.32 g/mol), superoxide dismutase, and catalase. Eluted purified hemoglobin (64,458 g/mol) from the PD-column will be collected in another tube. The working solution of HbA is made with 40μM HbA at pH 7.4 in water.
