Human Plasma and Recombinant Hemopexins: Heme Binding Revisited

Plasma hemopexin (HPX) is the key antioxidant protein of endogenous clearance pathway that limits deleterious effects heme released from hemoglobin and myoglobin (the term “heme” used in this article to denote both ferrous ferric forms). During intra-vascular hemolysis, partitioning lipid increases as plasma concentration HPX declines. Therefore, development a replacement therapy during high stress could be relevant intervention for hemolytic disorders. A logical approach enhance yield involves recombinant production strategies human cell lines. The present study focuses on biophysical assessment binding (rhHPX) produced Expi293FTM (HEK293) system. In report, we examine rhHPX comparison with using systematic analysis structural functional characteristics related binding. Analysis by UV/Vis absorption spectroscopy, circular dichroism (CD), size-exclusion chromatography (SEC)-HPLC, catalase-like activity demonstrated similarity fractionated plasma. particular, titration apo-protein(s) was performed first time wide range concentrations model HPX–heme interactions approximate physiological conditions (from extremely low more than two-fold molar excess over protein). CD data showed an induced bisignate Soret band pattern typical versions at heme-to-protein ratios further dependent amount protein-bound extent arising opposite couplet results complete inversion observed pattern. generated suggest one site rhHPX. Furthermore, our provides useful analytical platform detailed characterization potentially novel fusion constructs.