Scalable production and biophysical characterization of an enzyme cocktail derived from human red blood cells

Red blood cells (RBCs) play a critical role in oxygen and carbon dioxide transport, which is facilitated by RBC-encapsulated hemoglobin (Hb) and carbonic anhydrase (CA). In addition, RBCs are constantly exposed to oxidative stress due to the intracellular reactive oxygen species (ROS) generated during Hb auto-oxidation. Antioxidant enzymes within RBCs, such as superoxide dismutase (SOD), catalase (CAT), and peroxiredoxin (Prx), counteract ROS generation to protect the RBC from oxidative stress. Therefore, this study presents a scaled-up method to extract an enzyme cocktail from lysed human RBCs, enriched with the major RBC enzymes with minimal Hb contamination. Using ethanol-chloroform precipitation and multiple biophysical analyses (SDS-PAGE, SEC-HPLC, MALDI-TOF, and LC-MS/MS), the RBC enzymes were successfully separated from Hb in the hemolysate. The purified enzyme cocktail exhibited minimal Hb contamination and retained a significant amount of CA, and antioxidative enzymes like SOD and CAT. Therefore, this scalable RBC enzyme purification method provides an efficient approach for isolating RBC enzymes with broad biomedical relevance.