The preparation of stroma-free hemoglobin by selective DEAE-cellulose absorption

“…Several groups have explored various methods for purifying Hb by anion exchange chromatography [21][22][23][24]. In the 1960's, Hb was purified using DEAE cellulose chromatography [21].…”

Section: Introductionmentioning

confidence: 99%

“…However, this technique suffered from very slow flow rates (∼ 80-100 mL per hour in a 0.9 cm diameter column) and the presence of highly toxic KCN in the buffer solutions. Cheung et al [23] improved on this method and prepared stroma-free hemoglobin by selective DEAE-cellulose absorption. Typical separations were completed in 11 hours, which was faster when compared to the crystallization method (4 days).…”

Section: Introductionmentioning

confidence: 99%

Preparation of ultrapure bovine and human hemoglobin by anion exchange chromatography

Sun

Palmer²

2008

Journal of Chromatography B

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Bovine and human hemoglobin (Hb) form the basis for many different types of Hb-based O 2 carriers (HBOCs) ranging from chemically modified Hbs to particle encapsulated Hbs. Hence, the development of a facile purification method for preparing ultrapure Hb is essential for the reliable synthesis and formulation of HBOCs. In this work, we describe a simple process for purifying ultrapure solutions of bovine and human Hb. Bovine and human red blood cells (RBCs) were lyzed, and Hb was purified from the cell lysate by anion exchange chromatography. The initial purity of Hb fractions was analyzed by SDS-PAGE. Pure Hb fractions (corresponding to a single band on the SDS-PAGE gel) were pooled together and the overall purity and identity assessed by LC-MS. LC-MS analysis yielded two peaks corresponding to the calculated theoretical molecular weight of the α and β chains of Hb. The activity of HPLC pure Hb was assessed by measuring its oxygen affinity, cooperativity and methemoglobin level. These measures of activity were comparable to values in the literature. Taken together, our results demonstrate that ultrapure Hb (electrophoresis and HPLC pure) can be easily prepared via anion exchange chromatography. In general, this method can be more broadly applied to purify hemoglobin from any source of RBC. This work is significant, since it outlines a simple method for generating ultrapure Hb for synthesis and/or formulation of HBOCs.

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“…Because of the long time required (4 days), crystallization has been replaced by chromatographic methods. Proteins other than hemoglobin, present in hemolysates, were separated by absorption on DEAE-cellulose (DEAE-52) at pH 7.5, using 0.01 mol=L sodium phosphate buffer, but no attempt was addressed to remove phosphoslipids [17]. In order to minimize hemoglobin auto-oxidation, saturation of hemoglobin solutions with carbon monoxide (CO) was suggested.…”

Section: Introductionmentioning

confidence: 99%

Stroma-Free Hemoglobin from Bovine Blood

Lima

Andrade

2007

Artificial Cells, Blood Substitutes, and Biotechnology

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Isolation and purification of bovine hemoglobin (HbBv) was carried out after reaction of whole blood with carbon monoxide. Washing/centrifugation steps were used to eliminate leukocytes, platelets, and plasma proteins. Hypotonic media and ultrasound radiation were used to lyse red blood cells. Lyse by ultrasound was shown to lead to solutions at the highest concentrations in HbBv, and the least concentrations in major phospholipids contaminants. Additional purification procedures were performed to remove membrane proteins and phospholipids. In the first case, proteins were denatured by thermal treatment, and filtered. To eliminate phospholipids, liquid chromatography was used with strong anion exchangers. Purity of HbBv was evaluated by normal phase high performance liquid chromatography (HPLC), electrophoresis, and size-exclusion HPLC.

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The preparation of stroma-free hemoglobin by selective DEAE-cellulose absorption

Cited by 21 publications

References 8 publications

The role of pH and its control on effective conjugation of bovine hemoglobin and human serum albumin

The role of pH and its control on effective conjugation of bovine hemoglobin and human serum albumin

Preparation of ultrapure bovine and human hemoglobin by anion exchange chromatography

Stroma-Free Hemoglobin from Bovine Blood

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