APPLICATION OF AQUEOUS 2-PHASE SYSTEM IN SEPARATION PURIFICATION OF STROMA FREE HEMOGLOBIN FROM ANIMAL BLOOD/

Aqueous two-phase system (ATPS) is suitable for treatment of biologica l materials. It has been traditionally employed in the separation of p roduct from fermentation broth, and in the extraction of large-molecul e proteins from colloid solution. The separation method based on ATPS features simple, rapid and continuous operation with low equipment cos t.A PEG/Salt aqueous two-phase system is studied, and developed for he moglobin (Hb) separation from animal bloods. Stroma free hemoglobin (S FHb) 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 pu rify SFHb. The purity of SFHb was analyzed by HPLC and IEF (silver sta ining). Large partition coefficients of Hb have been observed with pro per selection of PEG molecular weight, added concentrations of PEG and salt in each phase. The SFHb of very high purity which is free of oth er proteins (< 0.1% impurity) and phospholipids (< 2% impurities) may be obtained from human packed RBCs. A two-stage extraction design usin g ATPS separation concept is very simple and useful for purification o f SFHb. The method may be further developed to offer potent technology for hemoglobin separation from animal blood and future production ind ustry of blood substitutes.