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

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.

Mapping of hemoglobin in erythrocytes and erythrocyte ghosts using two photon excitation fluorescence microscopy

“…During the process of gradual decrease of ionic strength of the solution surrounding erythrocytes, they swell and hemoglobin molecules leak out, leaving intact erythrocyte membranes. 13,17,18,29 Preparation of ghosts was followed by an extensive washing out procedure (to remove extracellular hemoglobin released from lysed erythrocytes). However, a small amount of hemoglobin, so called residual hemoglobin, always remains bound to membranes in resulting erythrocyte ghosts.…”

“…Based on the aforementioned wide applicability of erythrocytes in biomedical research and diagnostic tests, it is important to have a reliable microscopy method for single cell analysis, including spatial distribution of hemoglobin. Although phase contrast microscopy traditionally represents a fast method for tracking changes of erythrocytes during the gradual hypotonic hemolysis and appearance of erythrocyte ghosts, 17,13,18 it does not provide enough information for detailed morphological analysis. Unlike the phase contrast microscopy that visualizes all the changes of the index of refraction regardless of their origin, 2PE microscopy has high chemical selectivity, thus enabling the visualization of the distribution of certain chemical compounds.…”

“…This investigation opens the possibility to use 2PE microscopy as a tool for quality assessment of erythrocytes and erythrocyte membranes as a starting material in various biotechnological processes, such as the production of erythrocyte-based drug delivery systems 13,15 or isolation of hemoglobin. 18,29 Additionally, 2PE microscopy could help in revealing the ability of erythrocyte membranes, originating from different species, to bind different amounts of hemoglobin. Since various processes require various levels of purity, this information is important for the selection of raw material.…”

Maltose-mediated long-term stabilization of freeze- and spray- dried forms of bovine and porcine hemoglobin

“…In any slaughter, 4 to 5 L of blood per 100 kg of bovine/porcine mass could be obtained. 4,5 Knowing that 1 L of bovine blood contains 110 g of hemoglobin 6 and 0.4 g of heme iron, it was calculated that in a technological process with a yield of 70 % from blood of only one cattle, with an average weight of 500 kg, it is possible to isolate 1.7 kg of hemoglobin protein, 7 containing 6 g of heme iron. Unfortunately, slaughterhouse blood is mainly discarded and treated as a waste exposing a high pollutant capacity.…”

Biomembranes from slaughterhouse blood erythrocytes as prolonged release systems for dexamethasone sodium phosphate

“…Particularly, erythrocytes from slaughterhouse blood should be taken into consideration as a high amount is produced on a daily basis. Beside some reports on hemoglobin isolation and its enzymatic modifications, other applications of cellular fractions of bovine and porcine slaughterhouse blood, such as erythrocyte ghosts, are scarce . The erythrocyte membranes have been successfully isolated from bovine and porcine slaughterhouse blood by a pilot‐scale optimized gradual hypotonic hemolysis in our recent study, and the ghosts were characterized in terms of their biochemical and morphological properties.…”