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Isolation of Hemoglobin from Bovine Erythrocytes by Controlled Hemolysis in the Membrane Bioreactor

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Abstract

In this work, we describe an optimized procedure based on gradual hemolysis for the isolation of hemoglobin derived from bovine slaughterhouse erythrocytes in a membrane bioreactor. The membrane bioreactor system that provided high yields of hemoglobin (mainly oxyhemoglobin derivate) and its separation from the empty erythrocyte membranes (ghosts) was designed at a pilot scale. Ten different concentrations of hypotonic media were assessed from the aspect of the extent of hemolysis, hematocrit values of the erythrocyte suspensions, cell swelling, and membrane deformations induced by decreased salt concentration. Effective gradual osmotic hemolysis with an extent of hemolysis of 88% was performed using 35 mM Na-phosphate/NaCl buffer of pH 7.2–7.4. Under these conditions most of the cell membranes presented the appearance of the normal ghosts under phase contrast microscope. The hemoglobin purity of >80% was confirmed by SDS-PAGE. Kinetic studies showed that maximal concentration of hemoglobin was reached after 40 min, but the process cycle at which recovery of 83% was achieved lasted for 90 min. The dynamics of both steps, (1) transport through the membrane of erythrocytes during process of hemolysis and (2) transport through the reactor filters, were evaluated.

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Acknowledgements

This research was funded by grants III46010 and E!4486 from the Ministry of Science and Technological Development, Republic of Serbia. The authors thank to Dr Ivana Klun for the assistance in microscopy and image analysis.

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Authors and Affiliations

  1. Department of Chemical Engineering, Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11000, Belgrade, Serbia

    Radoslava Stojanović, Verica Manojlović, Marija Dević & Branko Bugarski

  2. Institute for Medical Research, University of Belgrade, Dr Subotica 4, POB 39, 11129, Belgrade 102, Serbia

    Vesna Ilić & Diana Bugarski

Authors
  1. Radoslava Stojanović
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  2. Vesna Ilić
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  3. Verica Manojlović
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  4. Diana Bugarski
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  6. Branko Bugarski
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Correspondence to Branko Bugarski.

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Stojanović, R., Ilić, V., Manojlović, V. et al. Isolation of Hemoglobin from Bovine Erythrocytes by Controlled Hemolysis in the Membrane Bioreactor. Appl Biochem Biotechnol 166, 1491–1506 (2012). https://doi.org/10.1007/s12010-012-9543-9

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