Recognition and Separation of Hemoglobin Variants via Molecularly Imprinted Polymers

Sammanfattning: Hemoglobin (Hb) is the predominant protein in red blood cells with allosteric regulation mechanisms for delivering oxygen between the lungs and tissues. After the 1980s, concentrated research on modified Hb emerged because of the necessity for the development of a blood substitute (oxygen carrier). Several purification procedures and genetic/chemical modifications of Hb have been undertaken by both industry and academia to produce viable Hb-based oxygen carriers (HBOCs). In this thesis, molecular imprinting technique has been applied to characterize, recognize and purify different Hb variants. Molecular imprinting is one of the most efficient methods to prepare polymer materials bearing pre-designed molecular recognition sites, which are complementary to the template in their size, shape and spatial arrangement of the functional groups. By combining Pickering emulsion polymerization and surface imprinting, highly cross-linked Hb-imprinted polymers with recognition sites on the surface were synthesized. The obtained molecularly imprinted polymers (MIPs) possessed high selectivity and could be applied as an efficient chromatography resin to selectively recognize and purify different Hb variants, such as adult Hb (HbA), fetal Hb (HbF), fusion HbF (fHbF) and Hb mutants. From MIP column, we also identified that E. coli glyceraldehyde 3-phosphate dehydrogenase (GAPDH) could especially interact with HbF that will further interfere with the regular E. coli metabolism and indicated the importance to carefully control cell metabolism when optimizing Hb production in heterologous systems.The possibility of using MIPs as Haptoglobin (Hp) mimics in vitro was also studied. Hb is safe and inert within the confinement of the RBCs but becomes reactive and toxic upon intravascular hemolysis. When red cells are lysed in vivo, Hp binds to free Hb instantly and prevent free Hb‐induced vascular dysfunction or injury. We demonstrated that MIPs, as a Hp mimic, could reduce the intrinsic oxidative toxicity associated with Hb. Furthermore, to facilitate the evaluation of the biophysical properties of HbF both in cell-free environments as well as in biological test systems, we have developed fluorescent Hb (GFP-fHbF) by genetic linkage of fHbF with the green florescent protein (GFP) at the DNA level. It was also applied to evaluate the efficiency of both HbA-imprinted and HbF-imprinted polymer beads. Unlike physical absorption of HbA on silica surface, the preparation of the HbF-imprinted polymers is achieved by covalently immobilizing HbF to silica nanoparticles as templates. Overall, our work opens up new possibilities of carefully designed MIPs for tailored protein purification, separation and analysis.