Application of aqueous two-phase systems in separation/purification of stroma free hemoglobin from animal blood.

Aqueous two-phase system (ATPS) is suitable for treatment of biological materials. It has been traditionally employed in the separation of product from fermentation broth, and in the extraction of large-molecule proteins from colloid solution. The separation method based on ATPS features simple, rapid and continuous operation with low equipment cost. A PEG/Salt aqueous two-phase system is studied, and developed for hemoglobin (Hb) separation from animal bloods. Stroma free hemoglobin (SFHb) was extracted from 200ml packed red blood cells by the two-stage aqueous two-phase system and subsequently was desalted and dialyzed by ultrafiltration. It took only 2.5hr to complete overall process to purify SFHb. The purity of SFHb was analyzed by HPLC and IEF (silver staining). Large partition coefficients of Hb have been observed with proper selection of PEG molecular weight, added concentrations of PEG and salt in each phase. The SFHb of very high purity which is free of other proteins (< 0.1% impurity) and phospholipids (< 2% impurities) may be obtained from human packed RBCs. A two-stage extraction design using ATPS separation concept is very simple and useful for purification of SFHb. The method may be further developed to offer potent technology for hemoglobin separation from animal blood and future production industry of blood substitutes.