The Purification and Comparative Analysis of Hemoglobin from Animal Bloods

Lee, C. J.; Kan, Pei; Chen, W. K.

doi:10.3109/10731199209119672

Cited by 3 publications

(3 citation statements)

References 6 publications

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“…Several methods have been developed to purify Hb from RBC lysate 8, 23–28. Currently practiced methods for Hb purification include extraction with organic solvent8 or separation by ion‐exchange chromatography 25, 29–31.…”

Section: Introductionmentioning

confidence: 99%

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Tangential flow filtration of hemoglobin

Palmer

Sun

Harris

2008

Biotechnology Progress

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in Wiley InterScience (www.interscience.wiley.com).Bovine and human hemoglobin (bHb and hHb, respectively) was purified from bovine and human red blood cells via tangential flow filtration (TFF) in four successive stages. TFF is a fast and simple method to purify Hb from RBCs using filtration through hollow fiber (HF) membranes. Most of the Hb was retained in stage III (100 kDa HF membrane) and displayed methemoglobin levels less than 1%, yielding final concentrations of 318 and 300 mg/mL for bHb and hHb, respectively. Purified Hb exhibited much lower endotoxin levels than their respective RBCs. The purity of Hb was initially assessed via SDS-PAGE, and showed tiny impurity bands for the stage III retentate. The oxygen affinity (P 50 ) and cooperativity coefficient (n) were regressed from the measured oxygen-RBC/Hb equilibrium curves of RBCs and purified Hb. These results suggest that TFF yielded oxygen affinities of bHb and hHb that are comparable to values in the literature. LC-MS was used to measure the molecular weight of the alpha (a) and beta (b) globin chains of purified Hb. No impurity peaks were present in the HPLC chromatograms of purified Hb. The mass of the molecular ions corresponding to the a and b globin chains agreed well with the calculated theoretical mass of the a-and b-globin chains. Taken together, our results demonstrate that HPLC-grade Hb can be generated via TFF. In general, this method can be more broadly applied to purify Hb from any source of RBCs. This work is significant, since it outlines a simple method for generating Hb for synthesis and/or formulation of Hbbased oxygen carriers.

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Section: Introductionmentioning

confidence: 99%

“…Aqueous‐phase extraction has also been used to purify Hb 23. In this method, Hb separation is achieved via a two‐step aqueous phase separation procedure.…”

Section: Introductionmentioning

confidence: 99%

Tangential flow filtration of hemoglobin

Palmer

Sun

Harris

2008

Biotechnology Progress

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“…Stroma free haemoglobin (SFHb) was extracted from human packed erythrocytes by the two-stage aqueous two-phase system and subsequently was desalted and dialyzed by ultrafiltration (Kan and Lee, 1994;Lee and Kan, 1995). The high purity of SFHb was obtained.…”

Section: Methodsmentioning

confidence: 99%

Effect of saturated/unsaturated phosphatidylcholine ratio on the stability of liposome-encapsulated hemoglobin

Huang¹,

Chung²,

Wu³

1998

International Journal of Pharmaceutics

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Swine hemoglobin as a potential source of artificial oxygen carriers, hemoglobin-vesicles

Sakai

et al. 2012

Artificial Cells, Nanomedicine, and Biotechnology

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Hemoglobin-based oxygen carriers (HBOCs) have been developed as a transfusion alternative and oxygen therapy. The Hb source is usually outdated donated human blood or cow blood obtained from cattle industries because of its abundance. This study examined the feasibility of using swine Hb ((S)Hb) for preparation of cellular-type HBOCs, hemoglobin-vesicles (HbV). Purification of (S)Hb from fresh swine whole blood was conducted with processes including carbonylation ((S)HbO(2) --> (S)HbCO), pasteurization (60 °C, 15 hours) and tangential flow ultrafiltration, with yield of 90%. Actually, differential scanning calorimetric analysis showed a denaturation temperature of (S)HbCO at 83 °C and assures its stability during pasteurization. Concentrated (S)HbCO together with pyridoxal 5'-phosphate (PLP) as an allosteric effector was encapsulated in phospholipid vesicles to prepare (S)HbV. After decarbonylation ((S)HbCO --> (S)HbO(2)), the oxygen affinity (P(50)) of (S)Hb changes mainly by PLP, and the influence of Cl(-) was small, in a manner similar to that of human Hb ((H)Hb). However, after encapsulation, vesicles of (S)HbV showed much lower oxygen affinity (higher P(50)) than (H)HbV did. Autoxidation of (S)HbV was slightly faster than (H)HbV. Although some differences are apparent in oxygen affinity and autoxidation rates, results clarified that (S)Hb is useful as a starting material for HbV production.

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The Purification and Comparative Analysis of Hemoglobin from Animal Bloods

Cited by 3 publications

References 6 publications

Tangential flow filtration of hemoglobin

Tangential flow filtration of hemoglobin

Effect of saturated/unsaturated phosphatidylcholine ratio on the stability of liposome-encapsulated hemoglobin

Swine hemoglobin as a potential source of artificial oxygen carriers, hemoglobin-vesicles

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