A continuous-flow high-yield process for preparation of lipid-free hemoglobin.
Hypotonic hollow-fiber dialysis of bovine red blood cells followed by ultrafiltration through 0.1-micron pore hollow fibers provides a simple method for isolation of lipid-free hemoglobin. Hemoglobin (Hb) isolated by comparative techniques were all contaminated with membrane stroma. HPLC analysis of Hb revealed a protein peak of 99.6% purity and sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis revealed a single band. The process requires hypoosmotic dialysis of bovine RBC to a final 160-180 mosmol/kg osmotic pressure. Additional reduction in osmotic pressure causes irreversible cell lysis which leads to lipid contamination of the Hb. Processing of 1/2 liter of packed red blood cells requires 4-5 h, resulting in an average of 90% hemoglobin recovery.
D000818:Animals; D002417:Cattle; D003956:Dialysis / Q000379:methods*; D006454:Hemoglobins / Q000302:isolation & purification*; D009997:Osmotic Pressure; D014462:Ultrafiltration / Q000295:instrumentation
D006454:Hemoglobins