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Preparation of Lipid‐Free Human Hemoglobin by Dialysis and Ultrafiltration
Author(s) -
Sheffield CL,
Spates GE,
Droleskey RE,
Green R.,
DeLoach JR
Publication year - 1987
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1987.tb00474.x
Subject(s) - hemoglobin , ultrafiltration (renal) , dialysis , chromatography , lysis , chemistry , osmotic pressure , sodium dodecyl sulfate , tonicity , red blood cell , sodium , osmotic concentration , biochemistry , surgery , medicine , organic chemistry
Dialysis of human red blood cells using a hypotonic solution and a commercial kidney dialysis unit followed by ultrafiltration through 0.1 micron pore hollow fibers provides an easily managed method for isolation of lipid‐free hemoglobin. High pressure liquid chromatography analysis of lipid‐free hemoglobin (LFHB) indicates 99–100% protein purity. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis demonstrated that LFHB migrates as a single band. The process requires hypoosmotic dialysis of human RBC to a final 119–139 (av 132) mosmol/kg osmotic pressure. Additional reduction in osmotic pressure results in irreversible cell lysis which results in lipid contamination of the hemoglobin. Processing one‐half liter of packed red blood cells requires 10 h, resulting in an average of 90% hemoglobin recovery.