Hemopexin Purification From Human Cohn Fraction IV Paste and Its Biophysical Characterization and Functional...

Hemopexin (Hpx) is an acute phase plasma protein that is responsible for sequestration and removal of cell-free heme with very high affinity (K_d < 1 pM). Hpx expression in liver cells is induced following an inflammatory event such as severe hemolysis. Therefore, plasma Hpx has potential clinical relevance due to its' ability to bind free heme, thus reducing lipid peroxidation and activation of inflammatory pathways in genetic anemias, such as sickle cell disease (SCD). This provides the rationale for purifying Hpx at high purity and at large scale. Starting from human Cohn fraction IV, we purified a protein cocktail using tangential flow filtration for use in this study as the starting material for purification of Hpx. Hpx was purified from the protein cocktail using immobilized metal ion affinity chromatography. SDS-PAGE and densitometric analysis showed that Hpx was purified to homogeneity and was approximately 99% pure. To gain further insight into Hpx activity and its' ligand binding properties, we employed comparative biophysical techniques such as UV-visible, circular dichroism, electron paramagnetic resonance and stopped flow spectroscopy. Finally we evaluated Hpx in a mouse model of SCD to test the ability of the novel material to attenuate tissue iron accumulation.