Purification and analysis of a protein cocktail capable of scavenging cell-free hemoglobin, heme, and iron.

Abstract

Hemolysis releases toxic cell-free hemoglobin (Hb), heme, and iron, which overwhelm their natural scavenging mechanisms during acute or chronic hemolytic conditions. This study describes a novel strategy to purify a protein cocktail containing a comprehensive set of scavenger proteins for potential treatment of hemolysis byproducts. Tangential flow filtration was used to purify a protein cocktail from Human Cohn Fraction IV (FIV). A series of in vitro assays were performed to characterize composition and biocompatibility. The in vivo potential for hemolysis byproduct mitigation was assessed in a hamster exchange transfusion model using mechanically hemolyzed blood plasma mixed with the protein cocktail or a control colloid (dextran 70 kDa). A basis of 500 g of FIV yielded 62 ± 9g of a protein mixture at 170 g/L, which bound to approximately 0.6 mM Hb, 1.2 mM heme, and 1.2 mM iron. This protein cocktail was shown to be biocompatible in vitro with red blood cells and platelets and exhibits nonlinear concentration dependence with respect to viscosity and colloidal osmotic pressure. In vivo assessment of the protein cocktail demonstrated higher iron transport to the liver and spleen and less to the kidney and heart with significantly reduced renal and cardiac inflammation markers and lower kidney and hepatic damage compared to a control colloid. Taken together, this study provides an effective method for large-scale production of a protein cocktail suitable for comprehensive reduction of hemolysis-induced toxicity.