Purification of bovine hemoglobin via fast performance liquid chromatography

Dimino, Michael L.; Palmer, Andre F.

doi:10.1016/j.jchromb.2007.05.040

Cited by 31 publications

(32 citation statements)

References 23 publications

(20 reference statements)

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“…The impurity fractions collected from peak 2 of Figure 1 show weak Hb bands, arising from individual alpha and beta subunits (M W ∼ 15 kDa), and strong impurity protein bands (M W ∼ 30 kDa). Other groups have also observed similar impurity bands [22,24]. Figures 3 and 4 show the chromatogram and SDS-PAGE of purified hHb, respectively.…”

Section: Oxygen Equilibrium Curvessupporting

confidence: 53%

“…It is interesting to note that in this work a much lower salt gradient was used to elute Hb off the column compared to other published reports [22,24]. This resulted in greater resolution of the Hb peak [22,24]. In all cases, the majority of Hb absorbed on the column was eluted off with the application of the linear gradient.…”

Section: Purification Of Hbmentioning

confidence: 87%

“…In several publications [22,24], a 20 cm column length was selected for Hb purification. However, in this study, a longer column (40 cm length) was used, therefore better Hb separation resolution was achieved.…”

Section: Purification Of Hbmentioning

confidence: 99%

“…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, in this method PEG has to be removed from the Hb fractions after purification. Dimino and Palmer [24] compared the absorption capacities of different anion exchange resins for Hb, and found that Q Sepharose XL had the highest Hb absorption capacity. This knowledge was used to purify Hb, which still contained several impurity proteins.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Oxygen Equilibrium Curvessupporting

confidence: 53%

Section: Purification Of Hbmentioning

confidence: 87%

Section: Purification Of Hbmentioning

confidence: 99%

“…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%

Section: Introductionmentioning

confidence: 99%

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Preparation of ultrapure bovine and human hemoglobin by anion exchange chromatography

Sun

Palmer²

2008

Journal of Chromatography B

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Silk fibroin/calcium alginate composite modifying supermacroporous molecularly imprinted membrane synthesis for high performance on recognizing bovine hemoglobin

Fan

Huang

Cheng

et al. 2022

J of Applied Polymer Sci

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Bio‐macromolecule hemoglobin (Hb) is a kind of iron‐rich metalloprotein that abundant in erythrocytes with various physiological properties. Separating Hb from bio‐matrix with high efficacy is crucial to Hb application but quite challenging. In this study, a supermacroporous molecularly imprinted membrane (MIM) was prepared with high performance on recognizing bovine hemoglobin (BHb). By using the silk fibroin and calcium alginate as mechanical modifiers, the mechanical strength of the prepared membrane was significantly improved to at around 0.25 MPa, and the supermacroporous structure in MIM was also formed. The MIM adsorption capacity of BHb was as high as 121.8 mg g−1 at the initial concentration of 0.5 mg mL−1. The MIM separation factor of BHb from BHb/BSA mixtures was as high as 7.34, and the relative imprinting factor was at around 4.0. The prepared MIM demonstrated the superiority and the valuable application prospect on specific separation of BHb.

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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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Purification of bovine hemoglobin via fast performance liquid chromatography

Cited by 31 publications

References 23 publications

Preparation of ultrapure bovine and human hemoglobin by anion exchange chromatography

Preparation of ultrapure bovine and human hemoglobin by anion exchange chromatography

Silk fibroin/calcium alginate composite modifying supermacroporous molecularly imprinted membrane synthesis for high performance on recognizing bovine hemoglobin

Tangential flow filtration of hemoglobin

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