Stroma-Free Hemoglobin from Bovine Blood

Stroma-Free Hemoglobin from Bovine Blood

Maria Celiana P. Lima and Cristina T. AndradeInstituto de Macromoleculas Professora Eloisa Mano, Universidade Federal do

Rio de Janeiro, Centro de Tecnologia, Rio de Janeiro, Brazil

Abstract: Isolation and purification of bovine hemoglobin (HbBv) was carriedout after reaction of whole blood with carbon monoxide. Washing=centrifugationsteps were used to eliminate leukocytes, platelets, and plasma proteins. Hypo-tonic media and ultrasound radiation were used to lyse red blood cells. Lyseby ultrasound was shown to lead to solutions at the highest concentrations inHbBv, and the least concentrations in major phospholipids contaminants.Additional purification procedures were performed to remove membrane pro-teins and phospholipids. In the first case, proteins were denatured by thermaltreatment, and filtered. To eliminate phospholipids, liquid chromatographywas used with strong anion exchangers. Purity of HbBv was evaluated by normalphase high performance liquid chromatography (HPLC), electrophoresis, andsize-exclusion HPLC.

Keywords: Bovine hemoglobin; Electrophoresis; High performance liquidchromatography; Ion exchange chromatography; Purification

1. INTRODUCTION

A significant number of research groups have been working on thedevelopment of oxygen carriers, so-called blood substitutes. These pro-ducts are designed to replace the respiratory function of hemoglobin incases of severe blood loss. They would offer clinical advantages overthe transfusion of red blood cells, including a long shelf life, minimal risk

The authors thank CNPq=MS (Proc. 50.5598=2004-3), CNPq (Proc.47.5320=2004-2), and FAPERJ (Proc. E-26=151.969=2004) for financial support.

Address correspondence to C. T. Andrade, IMA-UFRJ, Centro de Tecnologia,Bloco J, Avenida Jequitib�aa, n� 1450, Rio de Janeiro, RJ, Brazil 21945-970. E-mail:[email protected]

Artificial Cells, Blood Substitutes, and Biotechnology, 35: 431–447, 2007

Copyright Q Informa Healthcare

ISSN: 1073-1199 print/1532-4184 online

DOI: 10.1080/10731190701460333

431

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of transmitting infectious diseases (they could be sterilized), and no needof previous typing and cross-matching [1–8].

Currently, there are two major classes of oxygen carriers in phase IIIclinical trials, perfluorocarbon emulsions and hemoglobin-based oxygencarriers, which present different paradigms in relation to efficacy, safety,and production. In the case of hemoglobin-based oxygen carriers, theavailability of pure hemoglobin is a key factor in the preparation ofthe final products. The low supply of human hemoglobin from outdatedred blood cells consists in the most critical factor to its use in commercialproducts [9,10]. By contrast, bovine blood is abundant, and may be col-lected in slaughterhouses from controlled healthy animals. Moreover, inthe presence of physiological concentrations of chloride ions, bovinehemoglobin have an oxygen affinity similar to that of human hemoglobincompletely saturated by its allosteric effector 2,3-diphosphoglycerate [2].

Hemoglobin can transport oxygen outside the red blood cell. It iscomposed of two identical a and two identical b globin chains, eachbound to a heme group. This structure is stabilized by hydrogen bond-ings, van der Waals forces, intra-and intermolecular salt bonds. In sol-ution, tetrameric hemoglobin is in unfavorable equilibrium with its abdimers, which are easily filtered through the glomerulus. As a result,hemoglobin has a short half-life in circulation, and leads to tubuleobstruction and renal failure [11]. To prevent tetramer dissociation,hemoglobin has been chemically modified or encapsulated [12–14]. Priorto these procedures, residual stroma composed of lipids and membraneproteins, water-soluble proteins other than hemoglobin, and possiblepathogenic viruses should be removed [15,16].

The preparation of stroma-free hemoglobin has been described in theliterature, but few papers give detailed information on methods andquantitative evaluation on purity. Crystallization of human hemoglobinfrom sodium phosphate solution has been used for some years. Becauseof the long time required (4 days), crystallization has been replaced bychromatographic methods. Proteins other than hemoglobin, present inhemolysates, were separated by absorption on DEAE-cellulose (DEAE-52)at pH 7.5, using 0.01 mol=L sodium phosphate buffer, but no attemptwas addressed to remove phosphoslipids [17]. In order to minimizehemoglobin auto-oxidation, saturation of hemoglobin solutions with car-bon monoxide (CO) was suggested. This procedure was followed as thefirst step to obtain concentrated solutions from outdated human redblood cells [18]. Organic solvents were used for hemolysis and removalof stromata. Heating to 60–62�C led to denaturation of water-solubleproteins other than hemoglobin, which were removed by centrifugation[18]. A more complex methodology was described for large-scale pro-duction of hemoglobin from outdated human red blood cells [19]. For

432 M. C. P. Lima and C. T. Andrade

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the extraction of phospholipids from red blood cells or aqueous solutionsof hemoglobin, styrene-divinylbenzene commercial disks were used.Phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine,and sphingomyelin were recovered at 92% yield in average, whereas therecovery yield of phosphatidylserine was 65% [20]. More recently, bovinehemoglobin (HbBv) was purified by a two-step process [21]. In the first,ion-exchange chromatography was used to remove other proteins fromhemolysates. In the second step, lipids from the cell membrane were elimi-nated by hydrophobic interaction chromatography. Optimization of themethodology was achieved by adding poly(ethylene glycol) to the phos-phate buffer at pH 6.8 [21].

In a previous work by our group, proteins other than hemoglobinwere eliminated from lysed outdated human red blood cells, previouslytreated by CO gas, by thermal treatment at 60�C followed by filtration.Ion-exchange chromatography was used to remove residual phospholipidsfrom small volumes of hemolysates. High-performance liquid chromato-graphy and electrophoresis were performed to evaluate purity [22]. Inthe present work, bovine hemoglobin (HbBv) was isolated and purifiedfrom whole bovine blood. Using available techniques to evaluate purity,HbBv solutions were obtained without the presence of phospholipidsand proteins other than hemoglobin.

2. MATERIALS AND METHODS

2.1. Materials

Bovine blood was collected in an anticoagulant citrate=dextrose solutionand maintained at 5�C until purification procedures. The dye-bindingreagent used for albumin quantification (2.5 mM bromcresol in 0.82 Mlactic acid at pH 4 containing 30 mL=L of Tween 80), the stock solutionof bovine serum albumin at 0.4 g=L stabilized with sodium azide,Drabkin’s reagent and hemiglobincyanide (HiCN) were purchased fromDoles Reagentes (Goiania, Brazil). Phospholipid standards phosphati-dylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine(PC) and sphingomyelin (SM), bovine serum albumin and MW-SDS-70L Kit for Electrophoresis were purchased from Sigma Chemical Co.(St. Louis, MO, USA) and used as received. Acetonitrile and methyl alco-hol (HPLC grade) were provided by Vetec Quımica Fina Ltda. (Rio deJaneiro, Brazil).

Anion exchangers, AG MP-1 from BioRad Labs (Bromley, England)and Q Sepharose Fast Flow (Q-SFF) from Pharmacia Biotech(Wikstroms, Sweden) were used after purification and conditioning.

Stroma-Free Hemoglobin from Bovine Blood 433

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The AG MP-1 resin was washed with ethyl alcohol and with deionizedwater, treated with 0.1 N NaOH for 15 min and neutralized by successivewashings, and finally treated with 0.1 N HCl for 15 min and neutralizedby extensive washings.

All other reagents and solvents (PA grade) were supplied by VetecQuımica Fina Ltda. (Rio de Janeiro, RJ, Brazil) and used as received.

2.2. Isolation of Hemoglobin

Bovine blood (200 g) was submitted to carbonylation reaction with COgas under mild shaking for 180 s, to convert oxyhemoglobin (HbO2) tocarbonylhemoglobin (HbCO) [18], and centrifuged at 1000 g at 25�Cfor 20 min in a Hermle Centrifuge model Z 383 K (National Labnet Com-pany Inc., New Jersey, USA) to eliminate leukocytes, platelets and someplasma proteins other than hemoglobin. The supernatant was discarded.The resulting suspension was washed with an equal weight of isotonic sal-ine solution (0.9% NaCl, w=v), and centrifuged at 1000� g for 20 min.The washing=centrifugation procedure was repeated three other times.

To verify the efficiency of the washing=centrifugation process, eachsupernatant was analyzed at 626 nm in a Thermolyne Turner spectro-photometer, model SP-870 (Dubuque, IA, USA) according to the brom-cresol green method at pH 4 [23], using a calibration curve prepared withdilute solutions of bovine serum albumin.

Hemolysis was performed in water, in 1 mM NaCl, and in 10 mMTris=HCl buffer at pH 7.4, using 1:1 and 1:2 (w=w) ratios of bovinered blood cells concentrate=hypotonic solution at 8�C for 24 h. Hemolysiswas also carried out by sonication at 8�C for 5 min with a 750 W ColeParmer Processor (Vernon Hills, IL, USA), equipped with a standardpin of 13 mm diameter at 40% amplitude.

After hemolysis, the suspension of lysed cells was submitted to heat-ing at 60�C for 1 h in a water bath under stirring in the dark, and centri-fuged at 2000� g for 40 min. The bottom layer was discarded. Theresulting hemoglobin solution was filtered through 0.22 mm Milliporemembranes.

2.3. Purification of Hemoglobin by Ion Exchange Chromatography

2.3.1. Small Volumes

A Flash 12i chromatography system, purchased from Biotage, Divisionof Dyax Corporation (Charlottesville, VA, USA) was used. Polyethylene


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columns of 75 mm� 12 mm were prepared by allowing 8.5 mL of theanion exchangers AG MP-1 or Q-SFF to settle under gravity. Mixedcolumns were also prepared by packing equal amounts of both resinssuccessively. Prior to use, each column was equilibrated at room tempera-ture and 1.0 Pa with 10 mM Tris=HCl pH 7.4 for approximately 30 min.Hemoglobin lysates (1 mL) were eluted with the same buffer at ratesthat varied (in the range 0.05–0.68 mL=min). Fractions of 1 mL werecollected and analyzed by spectrophotometry to determine hemoglobinconcentration.

2.3.2. Liquid Column Chromatography

Higher volumes of lysates (100 mL) were purified by liquid column chro-matography. A glass column 55 mm in diameter and 200 mm in heightwas used with 23 mL of the anion exchangers AG MP-1 or Q-SFF, whichwere settled under gravity. Mixed columns were also prepared by packingequal amounts of both resins successively. After equilibration with10 mM Tris=HCl pH 7.4 at room temperature for 12 h, hemoglobinlysates (100 mL) were eluted with the same buffer. Fractions of 5 mL werecollected and analyzed by spectrophotometry to determine hemoglobinconcentration.

2.4. Determination of Hemoglobin Concentration

Hemoglobin concentrations were determined by the hemiglobincyanide(HiCN) method [24]. Hemoglobin lysates and purified hemoglobin solu-tions (20 mL) were mixed with 5 mL of 1:100 diluted Drabkin’s reagent,homogeneized for 3 min and analyzed by absorption spectrophotometryat 540 nm. In each case, the hemoglobin concentration was determined inrelation to a calibration curve, prepared with diluted solutions of stan-dard HiCN.

2.5. Characterization of Isolated and Purified Bovine Hemoglobins

2.5.1. Extraction of Phospholipids

Phospholipids were extracted from the hemolysates and from hemoglo-bin solutions after purification by anion exchange chromatography,according to the literature [25]. To 20 mL of a solution prepared by dilut-ing 2 g of hemoglobin lysate with 25 mL of deionized water, 50 mL methylalcohol and 25 mL methylene chloride were added. After stirring for


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10 min, 25 mL of methylene chloride and 25 mL of 2 M KCl were added.The resulting mixture was stirred for 10 min and transferred to adecantation funnel. The lower layer was collected and dried at 40�C underlow pressure. In the case of purified hemoglobin solutions, 1 g was takenfrom the two most concentrated fractions, to which 10 mL of deionizedwater were added, and the procedure described above was followed.The residue obtained by drying the lower layer was redissolved in 1 mLchloroform, filtered in poly(trifluorethylene) 0.22 mm membranes andused to analyze the presence of phospholipids by high performance liquidchromatography.

2.5.2. High Performance Liquid Chromatography (HPLC) of ResidualPhospholipids

Normal phase HPLC was carried out with a Pharmacia LKB-HPLCpump model 2248 from Pharmacia-Biotech (Uppsala, Sweden), equippedwith a HP 3396 Series II integrator from Hewlett Packard (Palo Alto,CA, USA). Separations were performed on a stainless-steel column(250 mm� 4.6 mm i.d.), packed with porous silica 100 A in diameter,from Phenomenex, (Montgomeryville, PA, USA) at 30�C and a constantflow rate of 1.0 mL=min. Solutions of the standard phospholipids phos-phatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcho-line (PC) and sphingomyelin (SM) were prepared in chloroform at1.5 g=L. A stock solution of mixed standard phospholipids was preparedat 144 mg=mL PS, 10 mg=mL PE, 120 mg=mL PC, and 300 mg=mL SM con-centrations, and used to quantify residual phospholipids from hemoly-sates and purified hemoglobin solutions. The samples were appliedwith a Hamilton syringe via a 20 mL Rheodyne 7125 injector (Cotati,CA, USA) and eluted with acetonitrile=methyl alcohol=phosphoric acidin 900:95:5 volume ratio [18,26]. The same solvent mixture was used asmobile phase for the analysis of phospholipids extracted from hemoglo-bin experimental samples. The elution was monitored with a UV-VisShimadzu model SPD-10AV from Shimadzu Scientific Instruments(Columbia, MD, USA), set at 210 nm.

2.5.3. Electrophoresis

Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE)was carried out in a single-sided vertical Owl Scientific Inc. system,model P81 (Woburn, MA, USA), equipped with a ElectrophoresisPower Supply E 835 from Consort nv (Turnhout, Belgium), at 2 W.Gels were stained in Comassie blue G-250 and destained in acetic


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acid=ethyl alcohol aqueous solution. The experiments were performedaccording to the procedure of Laemmli. Samples at 1 g=L concentrationwere prepared by heat denaturation at 100�C for 5 min in a buffer con-taining 1.0 mL 1 M Tris=HCl pH 6.8, 4 mL deionized water, 1.6 mLSDS at 10% (w=v), 0.8 mL glycerol at 87% (v=v), and 0.2 mL bromphe-nol blue at 0.05% (w=v). Hemoglobin and MW-SDS-70L solutions(10 mL) were applied to each lane of the gel and processed at 30 mA,120 V, for 120–180 min. MW-SDS-70L markers are composed of bovinea-lactalbumin (MW�14,200), bovine trypsinogen (MW�24,000), bov-ine carbonic anhydrase (MW�29,000), glyceraldehyde-3-phosphate(MW�36,000), and bovine albumin (MW�66,000).

2.5.4. Size Exclusion HPLC of Purified Hemoglobin

Normal phase HPLC was carried out with the same equipmentdescribed in item 2.6.2, on a Biosep Sec S 3000 stainless-steel column(300 mm� 7.0 mm i.d.), packed with 5 mm porous silica 100 A in dia-meter, from Phenomenex (Montgomeryville, PA, USA) at room tem-perature, and a constant flow rate of 0.7 mL=min. The hemoglobinsample was dissolved in 0.1 M Tris=HCl pH 7.4 with 0.2 M MgCl2 at0.1% concentration. The same solvent mixture was used as mobilephase for the analysis. Elution was monitored with the same UV-Visequipment, set at 280 nm.

3. RESULTS AND DISCUSSION

Carbonylation of whole bovine blood was performed as the first step ofthe purification methodology. This procedure was followed to takeadvantage of the heat-stability of carbonylhemoglobin [27], and to facili-tate the removal of other proteins by heat-induced denaturation and fil-tration in a later stage. After centrifugation and elimination of thesupernatant, the suspension consisting of red blood cells (RBC) waswashed with 0.9 M NaCl, and centrifuged four times. Every cycle ofwashing=centrifugation was accompanied by quantification of albumin(in the supernatant), by spectrophotometry at 626 nm. Figure 1 showsthe decreasing concentration of albumin, from approximately 0.8 g=dLto undetectable levels.

As described in the experimental section, hemolysis was performedin hypotonic media and by ultrasound radiation. Water, 1 mM NaCl,and 10 mM Tris=HCl buffer pH 7.4, at 1:1 and 1:2 (w=w) ratios ofRBC=hypotonic solution were used. In all cases, hemolysates were free


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from blood cells, as visualized by optical microscopy. As expected,hemolysis by sonication gave rise to the most concentrated hemoglobinsolution (23.09� 3.60 g=dL), compared to hypotonic media in 1:1 ratio,which led to hemoglobin solutions in the range 14.24� 0.002 g=Lto 15.81� 0.005 g=L. Since prolonged standing in a diluted solution isknown to favor hemoglobin oxidation [28], this method of hemolysisseems adequate mainly when stock solutions are produced to be storedfor further uses.

Heat treatment at 60�C for 1 h was carried out to denature proteinsother than hemoglobin. Centrifugation and filtration were used to removecellular debris and denatured protein contaminants. Anion exchangechromatography was chosen to eliminate residual phospholipids.

Ion exchangers are the most widely used types of stationary phasesfor separation and purification of proteins. The relatively mild bindingand elution conditions allow high recovery with intact biologicalactivity. For small volumes, the Flash 12i chromatography system wasused with AG MP-1, Q-SFF and two layers of each resin as stationarymedium. Both AG MP-1 and Q-SFF resins are strong anion exchangers.In all cases, elution was carried out with 10 mM Tris=HCl pH 7.4. Theobjective was to adsorb lipids from the cell membrane and enable hemo-globin to flow through the exchange resins. Since HbBv is a globularprotein 55 A in diameter, and the pore dimensions of macroporousresins are larger than 100 A, HbBv can partially pass through the poresunder the applied pressure. Nevertheless, the red color of HbBv couldbe seen during the operation from outside of the cartridge, as well as

Figure 1. Average albumin concentration in supernatants after each washing=centrifugation procedure.


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outside of the glass column, and no significant retention of the proteinwas observed.

At pH 7.4, HbBv eluted at lower volumes on AG MP-1 stationaryphase, and the phospholipids were retained on the top of each column.Chromatograms for hemoglobin eluates that had been lysed in water,in 1 mM NaCl, in 10 mM Tris=HCl pH 7.4, and by ultrasound radiationwere obtained. As an example, Figure 2 shows the chromatograms ofHbBv, previously hemolysed in water at 1:2 (w=w) ratio of RBC=water,on AG MP-1, Q-SFF, and on double layers of both resins at pH 7.4 usingTris=HCl buffer as the mobile phase. Only one peak was obtained with areasonable width.

Higher volumes of purified HbBv were obtained by liquid columnchromatography. In this case, ultrasound radiation was the methodchosen to lyse RBC. Table 1 shows concentrations of HbBv determined

Figure 2. Average elution curves for the hemoglobin solution previouslyhemolysed in water at 1:2 ratio (w=w) of RBC=water; purified with AG MP-1 (�),Q-SFF (

4

), and AG MP-1=Q-SFF layers (&). Points represent average valuesfrom analyses in triplicate.

Table 1. HbBv concentrations determined for the most concentrated eluates pre-viously lysed by sonication, after purification by liquid column chromatographyon AG MP-1, Q-SFF, and AG MP-1=Q-SFF layers

Purified HbBv (g=dL)Isolated HbBv

(g=dL) AG MP-1 Q-SFF AG MP-1=Q-SFF layers

Sample 23.09� 3.60 12.59� 4.51 7.74� 2.33 16.69� 2.34


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for the most concentrated eluates. Differences in concentrations seem torely on the physical properties of the resins, whereas AG MP-1 is formedby solid beads that favor eluent and eluate phase separation, Q-SFF resinis a gel, in which the eluent is easily mixed with the eluate.

Normal phase HPLC with UV detection has been used to quantifythe main phospholipids from the red cell membrane [18,22,26]. To accessHbBv purity in relation to the presence of residual phospholipids, thistechnique was used with organic extracts, before and after anionexchange chromatography. Figure 3 shows HPLC chromatograms ofphospholipids markers, and phospholipids extracted from isolated hemo-globin solutions obtained by hemolysis in hypotonic media at 1:1 ratio ofRBC=hypotonic medium, and by ultrasound. The lowest limit of sensi-tivity of the HPLC method for the detection of PS and PE was0.01 mg=dL. For the detection of PC and SM, the lowest limits of sensi-tivity were 0.03 mg=dL, and 0.20 mg=dL, respectively. For the standardssolutions, elution times of 4.16, 5.30, 10.00, and 15.20 min were observedfor PS, PE, PC, and SM, respectively. SM appears as a double peakbecause consists of diastereomers [29].

As reported previously for human hemolysates, two other peaksdeserve attention. These peaks, denoted as U1 and U2, were attributedto diacylglyceride products resulting from degradation of phospholipids[26]. The first peak was detected around 6.57 min for every hemolysate.Peak U2 was detected at 7.94 min for the sample hemolysed by ultra-sound. Table 2 shows an estimation of phospholipids concentrationsdetected by HPLC for hemolysates in water, saline, and buffer solutions,and hemolysates produced by sonication. Hemolysis by sonication led tothe lowest concentrations of the main phospholipids, as observed beforefor human RBC [22]. As observed in Table 2, phospholipids compositionin hemolysates follows the same order as in bovine blood [30], SM beingthe most abundant.

Additional purification of hemolysates on anion exchangers elimi-nated residual PS, PE, PC, and SM, according to HPLC chromatograms.No peak was observed at the elution times corresponding to these phos-pholipids. For example, in Figure 4, HPLC chromatograms of organicextracts from water (1:1 ratio RBC=water) and ultrasound hemolysates,which had been additionally purified by liquid column chromatographyon AG MP-1, Q-FF and AG MP-1=Q-FF resins, revealed only one peakat the elution time of U1.

After storage for 60 days at �18�C, organic extracts from ultra-sound hemolysate samples, before and after additional purification, wereanalyzed by normal phase HPLC (Figure 5). In the case of the hemolysate,as shown in Table 3, and in comparison with the results shown in Table 2,the concentrations of PS, and SM decreased, whereas the concentration of


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Figure 3. Normal phase HPLC chromatograms for phospholipids from a sol-ution of commercially available markers (a); a water hemolysate (1:1 ratio ofRBC=water) (b); a saline hemolysate (1:1 ratio of RBC=1 mM NaCl) (c); a bufferhemolysate (1:1 ratio of RBC=10 mM Tris=HCl pH 7.4) (d); an ultrasound hemo-lysate (e).


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PE remained constant. As for PC, after storage, its concentrationdecreased to the point of not being detected. As for the diacylglyceride pro-ducts U1 and U2, a decrease in the intensity of the U1 peak was observed,whereas U2 became a major peak (Figure 5a). Peak U1 was completelyeliminated in the chromatogram, after elution of the hemolysate on theQ-SFF resin (Figure 5b). These results corroborate the hypothesis ofdegradation of stromal phospholipids with storage [26].

Analyses by SDS-PAGE electrophoresis were carried out for hemoly-sates and purified HbBv solutions. In Figure 6, SDS-PAGE analyses forthe hemolysate obtained by sonication (lane 2), and corresponding HbBvsamples purified on AG MP-1 (lane 3), Q-SFF (lane 4) and AG MP-1=Q-SFF (lane 5) resins were compared with the solution of mixed standardsprepared with Sigma MW-SDS-70L markers (lane 1). The hemolysate andthe purified samples migrated as a single band at approximately 14 kDa,which reveals the presence of no protein contaminants.

The presence of only one peak eluted at 13.5 min from size-exclusionHPLC (Figure 7) indicates recovery of the hemoglobin tetramer and elimin-ation of other proteins, such as a-, and b-spectrin, with higher molar masses.

CONCLUSION

Compared with hypotonic media, sonication can be indicated as a methodfor lysing red blood cells. The advantages of sonication are related to the

Table 2. Phospholipids concentrations in hemolysatesa, determined by normalphase HPLC

Phospholipids concentrations (mg=g HbBv solution)

Sampleb PS PE PC SM

Water hemolysate 31.39� 3.89 4.57� 0.28 20.02� 3.03 54.96� 3.07Saline hemolysate 97.07� 7.24 14.87� 1.88 77.39� 4.76 93.68� 0.18Buffer hemolysate 126.89� 11.24 21.21� 1.14 97.63� 9.39 137.48� 4.98Ultrasound hemolysate 23.46� 2.69 1.66� 0.48 3.71� 1.09 36.87� 0.91

aPS, phosphatidylserine; PE, phosphatidylethanolamine; PC, phosphatidylcho-line; SM, sphingomyelin; HbBv, bovine hemoglobin.

bWater hemolysate, hemoglobin solution isolated from bovine blood afterhemolysis in 1:1 (w=w) RBC=water; Saline hemolysate, hemoglobin solution iso-lated from bovine blood after hemolysis in 1:1 (w=w) RBC=1 mM NaCl; Bufferhemolysate, hemoglobin solution isolated from bovine blood after hemolysis in1:1 (w=w) RBC=10 mM Tris=HCl pH 7.4; Ultrasound hemolysate, hemoglobinsolution isolated from bovine blood after hemolysis by sonication.


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recovery of bovine hemoglobin solutions at higher concentrations, with alower concentration of the major phospholipids from the cell membrane.Thermal treatment of hemolysates was efficient to denature other proteinsthan hemoglobin, previously transformed into carbonylhemoglobin byreaction with carbon monoxide gas. Denaturing polyacrylamide gel

Figure 4. Normal phase HPLC chromatograms of phospholipids from a waterhemolysate submitted to additional purification by liquid chromatography onAG MP-1 (a); Q-SFF (b); AG MP-1=Q-SFF resins (c); an ultrasound hemolysatesubmitted to additional purification by liquid column chromatography on AGMP-1 (d); Q-SFF (e); AG MP-1=Q-SFF layers (f).


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electrophoresis revealed the presence of hemoglobin alone. Liquidchromatography on strong anion exchangers carried out with 10 mMTris=HCl pH 7.4 at room temperature removed residual phospholipids,as shown by UV-monitored high performance liquid chromatographyof organic extracts. Using sonication as the hemolysis method, the entire

Figure 5. Normal phase HPLC chromatograms of phospholipids from an ultra-sound hemolysate (a), and an ultrasound hemolysate additionally purified on theQ-FF resin (b), after storage for 60 days at � 18�C.

Table 3. Phospholipids concentrations in the ultrasound hemolysate after beingstored for 60 days at �18�C

SamplePS

(mg=mL)PE

(mg=mL)PC

(mg=mL)SM

(mg=mL)

Ultrasound hemolysatea 7.29� 2.41 1.66� 0.45 ndb 34.51� 0.25

aUltrasound hemolysate, hemoglobin solution isolated from bovine blood afterhemolysis by sonication.

bnd, nondetected.


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Figure 7. Size exclusion HPLC analysis of a HbBv sample lysed by sonicationand purified by liquid column chromatography on the Q-FF resin.

Figure 6. SDS-PAGE electrophoresis analyses for the hemolysate obtained bysonication (lane 2), and corresponding HbBv samples purified by liquid columnchromatography on AG MP-1 (lane 3), Q-SFF (lane 4), and AG MP-1=Q-SFFlayers (lane 5), in comparison with the standard protein solution (lane 1).


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process takes 15 h, and produces purified bovine hemoglobin at 16.7 g=dLconcentration.

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Documents

Stroma-Free Hemoglobin from Bovine Blood