Purification of diverse hemoglobins by metal salt precipitation

Accepted Manuscript Purification of Diverse Hemoglobins by Metal Salt Precipitation Devon Zimmerman, Jack Dienes, Osheiza Abdulmalik, Jacob J. Elmer PII: DOI: Reference:

S1046-5928(15)30056-5 http://dx.doi.org/10.1016/j.pep.2015.09.006 YPREP 4781

To appear in:

Protein Expression and Purification

Received Date: Revised Date: Accepted Date:

25 August 2015 5 September 2015 5 September 2015

Please cite this article as: D. Zimmerman, J. Dienes, O. Abdulmalik, J.J. Elmer, Purification of Diverse Hemoglobins by Metal Salt Precipitation, Protein Expression and Purification (2015), doi: http://dx.doi.org/10.1016/j.pep. 2015.09.006

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Purification of Diverse Hemoglobins by Metal Salt Precipitation Devon Zimmermana, Jack Dienesa, Osheiza Abdulmalikb, Jacob J. Elmera a – Villanova University, 800 East Lancaster Avenue, Villanova, PA 19085 b – Division of Hematology, Abramson Building, The Children's Hospital of Philadelphia, 34th St. & Civic Center Blvd, Philadelphia, PA 19104

Corresponding author: Jacob J. Elmer 217 White Hall, Department of Chemical Engineering 800 East Lancaster Avenue, Villanova, PA 19085 [email protected] (610)519-3093

Abstract Although donated blood is the preferred material for transfusion, its limited availability and stringent storage requirements have motivated the development of blood substitutes. The giant extracellular hemoglobin (aka erythrocruorin) of the earthworm Lumbricus terrestris (LtEc) has shown promise as a blood substitute, but an efficient purification method for LtEc must be developed to meet the potential large demand for blood substitutes. In this work, an optimized purification process that uses divalent and trivalent metal salts to selectively precipitate human, earthworm, and bloodworm hemoglobin (HbA, LtEc, and GdHb, respectively) from crude solutions was developed. Although several metal ions were able to selectively precipitate LtEc, Zn2+ and Ni2+ provided the lowest heme oxidation and highest overall yield of LtEc. In contrast, Zn2+ was the only metal ion that completely precipitated HbA and GdHb. Polyacrylamide gel electrophoresis (PAGE) analysis shows that metal precipitation removes several impurities to provide highly pure hemoglobin samples. Heme oxidation levels were relatively low for Zn2+-purified HbA and LtEc (2.4 ± 1.3% and 5.3 ± 2.1%, respectively), but slightly higher for Ni2+-purified LtEc (8.4 ± 1.2%). The oxygen affinity and cooperativity of the precipitated samples are also identical to samples purified with tangential flow filtration (TFF) alone, indicating the metal precipitation does not significantly affect the function of the hemoglobins. Overall, these results show that hemoglobins from several different species can be highly purified using a combination of metal (Zn2+) precipitation and tangential flow filtration. Highlights • • •

Earthworm hemoglobin is precipitated by a broad range of divalent and trivalent cations A purification method for hemoglobin and erythrocruorin using zinc has been optimized Three structurally different types of hemoglobin were successfully purified using this method

Keywords erythrocruorin, earthworm, hemoglobin, tangential flow filtration, precipitation, purification

Introduction According to the World Health Organization, over 80 million units of donated blood were transfused in 2009. However, while donated blood is a fairly common, effective, and safe treatment for hemorrhagic shock, it does have some significant disadvantages. For example, if blood donation rates continue to decrease while transfusion rates steadily increase, there may be a significant shortage of donated blood within the next 15 years [1,2]. Donated blood also has a limited shelf life of 42 days and must be constantly refrigerated [3]. Therefore, it is frequently unavailable in combat situations and remote areas that lack proper storage facilities. All of these issues have motivated the development of many different “blood substitutes” that can be transfused when donated blood is unavailable. The majority of blood substitutes are hemoglobin-based oxygen carriers (HBOC’s) [4]. Several HBOC’s based on human and bovine hemoglobins (HbA and bHb, respectively) have been developed, including Hemopure® (polymerized bHb, OPK Biotech) [5], Hemospan® (PEGylated HbA, Sangart) [6], and Oxyglobin® (polymerized bHb, Biopure) [7]. Many of these products showed promise in early clinical trials, but eventually failed Phase III clinical trials due to severe adverse reactions (e.g., vasoconstriction, hypertension, and stroke) that were attributed to nitric oxide scavenging and oxidation of the heme iron [4,8,9]. Several next generation HBOC’s have been designed to address these problems, including nanoparticles charged with hemoglobin [10,11,12], HemoTech (an ATP cross-linked bHb) [13], OxyVita (“zero-linked” high MW polymerized hemoglobin), and pPolyHb (cross-linked porcine hemoglobin) [14,15]. The extracellular hemoglobin (a.k.a. erythrocruorin, Ec) of the common earthworm Lumbricus terrestris (LtEc) and the marine lugworm Arenicola marina (AmEc) have also recently shown promise as novel blood substitutes. These Ec’s are large macromolecular complexes with naturally high molecular weights (~3.6 MDa) [16] that rival many of the current polymerized Hb’s (250 kDa-17 MDa) [17]. Preclinical studies with mice and hamsters have also shown that LtEc and AmEc do not elicit the harmful side effects (e.g., vasoconstriction) observed with previous polyHb’s [18,19]. In addition, studies with a hyper-responsive mouse strain (BP/2) showed that while ovalbumin elicits a strong immune response, AmEc showed almost no immune response relative to the vehicle control [19]. This lack of immune response has also been observed in clinical trials of polymerized bovine hemoglobins, suggesting that foreign hemoglobins from other organisms may be safe to use as blood substitutes [5,17,20]. Overall, these studies suggest that invertebrate hemoglobins may be promising candidates for blood substitute development, but further work will need to be done to fully determine the antigenicity of erythrocruorins. However, before any Hb or Ec can be considered a viable blood substitute, a suitable purification process must be optimized to provide large quantities of the Hb or Ec that can meet a large global demand. Many types of anion exchange (AEX) chromatography have been developed to purify mammalian Hb’s [21], but the small pore size of most AEX resins excludes relatively large Ec’s and severely reduces the Ec binding capacity of the resin. Consequently,

most Ec’s are purified by taking advantage of their unusually large size with size-exclusion chromatography (SEC) [22], ultracentrifugation [23,24], or tangential flow filtration (TFF) [18]. Alternatively, it has also been shown that HbA can be highly purified with immobilized metal (Zn2+) affinity chromatography (IMAC) or zinc precipitation [25,26]. The success of these methods is due to a high affinity Zn2+ binding site in the 2,3-diphosphoglycerate (2,3-DPG) binding site of HbA, which contains several histidine residues that can coordinate the Zn2+ ion [27]. There are also several additional histidine, glutamate, and aspartate residues on the surface of HbA that may contribute to Zn2+ binding. LtEc and other Ec’s do not bind 2,3-DPG, but they do have several metal binding sites (Ca2+, Cu2+, Zn2+) [28,29] and histidines on their surface [30], suggesting that Zn2+ (or other metals) may able to precipitate Ec’s as well. In this work, Zn2+ and many additional metal ions were screened (Figure 1) to determine which metals selectively precipitate HbA and LtEc. The most effective metal ions (Zn2+ and Ni2+) were then used to purify HbA, LtEc, and an additional hemoglobin from the invertebrate bloodworm Glycera dibranchiata (GdHb) [31]. Unlike LtEc and HbA, GdHb is present in two forms: a monomeric fraction (16-17 kDa) and a polymeric fraction with variable molecular weight (40~120 kDa) [32,33]. Despite these significant structural differences, metal precipitation was still able to provide highly pure HbA, LtEc, and GdHb solutions without any significant changes in O2 transport and minimal oxidation of the heme iron. These results show that metal precipitation is a quick and effective way to purify a wide variety of hemoglobins from diverse host organisms. Material and Methods Preparation of Crude Human Hemoglobin For each round of purification, 50 mL of donated human whole blood was purchased from Interstate Blood Supply (Memphis, TN). The blood was centrifuged at 10,000 g for 5 minutes at 4°C, then the resulting serum and white blood cell layers were removed. The packed RBC’s were then resuspended in the original volume (50 mL) of 20 mM phosphate buffered saline (PBS, pH 7.4) This process was repeated 3 times to remove as much serum as possible from the red blood cells. The remaining red blood cells were then lysed overnight at 4oC (16-18 hrs) in an equal volume of hypotonic 20 mM Tris buffer (pH 7.4). The solution was then centrifuged at 3,500 g for 15 minutes at 4°C to remove cell debris. The clarified red supernatant containing HbA was decanted and stored at 4°C until needed. Preparation of Crude LtEc For each round of purification, 100 Canadian night crawlers (Lumbricus terrestris) were purchased from Wholesale Bait Supply (Cincinnati, OH). Worms were rinsed with tap water to remove dirt and mucus and then homogenized in a blender (pulse mode for approximately ten seconds). The homogenate was then immediately centrifuged at 3,500 g for 30 minutes at 4°C.

The red supernatant was decanted into conical tubes and centrifuged at 15,000 g for 30 minutes at 4°C. The clarified red supernatant containing LtEc was then stored at 4oC until needed. Preparation of Crude GdHb For each round of purification, 30 bloodworms (Glycera dibranchiata) were purchased from Bloodworm Depot (Whitefield, Maine). Since preliminary experiments revealed that homogenization of bloodworms produces a solid clot, coelomic fluid was drained from each individual worm. The fluid was then centrifuged at 1,000 rpm for 1 minute at 4°C to separate the serum from the red blood cells (GdRBC’s). The serum was then decanted and ultrapure water was added in a 1:1 volume ratio to lyse the GdRBC’s. The solution was incubated at room temperature for 15 minutes to ensure complete lysis, then centrifuged at 3,500 g for 15 minutes at 4°C to remove cell debris. The clarified red supernatant containing GdHb was then decanted and stored at 4°C until needed. Metal Precipitation Screening Experiments Several divalent and trivalent metal ions were tested for the ability to precipitate HbA and LtEc, including zinc (Zn2+), nickel (Ni2+), chromium (Cr3+), copper (Cu2+), iron (II) (Fe2+), cobalt (Co2+), manganese (Mn2+), calcium (Ca2+), magnesium (Mg2+), iron (III) (Fe3+), vanadium (II) (V2+), cadmium (Cd2+), lead (Pb2+), and vanadium (III) (V3+). All metals were purchased as salts from Sigma (St. Louis, MO) and dissolved in 20 mM Tris buffer (pH 7.4) at stock concentrations of 1,000, 300, 100, 30, 10, 3, and 1 mM. In our preliminary precipitation screen, 500 µL of HbA or LtEc samples (50 or 500 µM heme concentration) that were previously purified by TFF [26] were mixed with 500 µL of each metal stock solution. The tubes were then centrifuged at 10,000 g for 5 minutes to detect precipitation and oxidation of the heme iron (indicated by a brown or black pellet). Top candidates (Zn and Ni) were defined as metals that induced complete precipitation of HbA and/or LtEc without significant oxidation. Purification Process An illustration of our purification process is shown in Scheme 1. Crude HbA, LtEc, and GdHb solutions (~500 µM heme) were combined in 1:1 volume ratio with a 100 mM ZnCl2 ( 20 mM Tris, pH 7.4) solution at room temperature and incubated for 5 minutes. The sample was mixed thoroughly and then centrifuged at 3,000 g for 15 minutes at 4°C to collect the precipitated hemoglobin. The HbA and LtEc samples precipitated completely under these conditions, so they produced a clear supernatant that was removed and concentrated ~1,00010,000 fold for polyacrylamide gel electrophoresis (PAGE) analysis. In contrast, the GdHb samples did not fully precipitate at any of the metal concentrations tested. However, addition of 1/10th volume of 10 mg/mL dithiothreitol (DTT), did provide almost complete precipitation of GdHb by Zn2+ (see supplementary material). In either case, the red supernatant (obtained without a reducing agent) and the clear supernatant (obtained with a reducing agent) were saved

for further analysis. Once the supernatant was removed from the HbA, LtEc, and GdHb samples, the red pellets were resuspended in 100 mM ethylenediaminetetraacetic acid (EDTA) (20 mM Tris, pH 7.4). The pellets were resuspended in a bottle containing a stir bar and wrapped with ice packs to protect the protein. The resuspended solutions were then centrifuged at 3,000 g for 15 minutes at 4°C to remove any remaining precipitates. The samples were then sterilized by filtering them through a 0.2 µm pore size TFF cartridge with a surface area of 790 cm2 (Spectrum Labs, Rancho Dominguez, CA). Excess metal, EDTA, and other protein impurities were then removed from the LtEc sample via ten rounds of diafiltration on two 500 kDa TFF filters as previously described [18]. In contrast, only metal and EDTA were removed from the GdHb and HbA samples via diafiltration on a 10 kDa TFF filter. In each round of diafiltration, the samples were concentrated to 50 mL, then diluted back to 500 mL with 20 mM Tris buffer, pH 7.0. After the final round of diafiltration, the oxidation level of each sample was measured and the samples were stored at -72oC until needed. All purification steps were performed at 4°C. PAGE Analysis Tricine gels were made at a concentration of 10% acrylamide, using the following recipe: 1.4 mL Millipore water, 4.3 mL Tricine Gel Buffer (3 M Tris, pH 8.45, 0.3% SDS), 1.3 mL 87% glycerol, 1.5 mL 2% polymerized acrylamide, 4.3 mL 30% acrylamide, 64 µL 10% ammonia persulfate, and 6.4 µL TEMED (tetramethylethylenediamine). The gels were run with a 0.2 M Tris, pH 8.9 anode buffer and a 0.1 M Tris, 0.1 M tricine, 0.1% SDS, pH 8.25 cathode buffer [34]. All samples containing hemoglobin were diluted to absorbances of A540 nm = 0.44 for LtEc and GdHb and A540 nm = 1.0 for HbA in Laemmli buffer with β-mercaptoethanol. Each gel was initially run at 30 volts for 10 minutes to separate excess salts, then the voltage was increased to 125 volts for approximately 2 hours. Native protein gels were at 8% acrylamide, using the following recipe: 2.6 mL 30% acrylamide, 7.30 mL 0.375 M Tris pH 8.8, 100 µL 10% ammonium persulfate, 10 µL TEMED. Each gel was run in a glycine running buffer (25 mM Tris, 192 mM) at 50 milliamps for 2 hours. Samples were all diluted to an absorbance of A540 nm ~ 0.22 in 62.5 mM Tris, pH 6.8, 25% glycerol, 1% bromphenol blue sample buffer. Cyanmethemoglobin Oxidation Assay Oxidation assays were conducted in 96 well plates with a Synergy HT Microplate Reader (BioTek, Winooski, VT) using the cyanmethemoglobin method [35]. Each sample was initially diluted by a factor of D1 until the absorbance at 630 nm (A630) of a 150 µL sample was between 0.1-1.0. This initial A630 reading was recorded as A1, then 20 uL of 10% KCN was added to the well and the A630 was recorded again as A2. These values were used to calculate the concentration of oxidized hemoglobin [Hb:Fe3+] in each sample using the following equation (λ1 = 0.45 cm, λ2 = 0.51 cm, ε1 = 3.7 cm-1 mM heme-1):





[ℎ:   ] =     −       (Eq. 1) 





In a separate reaction, another fresh sample was diluted by a factor of D2 until the absorbance at 540 nm (A540) of a 150 µL sample was between 0.3-1.0. 20 µL of 10% potassium ferricyanide K3[Fe(CN)6] was added to the sample and incubated at room temperature for 2 minutes, then 20 µL of 10% KCN was added to the sample and the A540 was recorded as A3. This value was then used to calculate the total concentration of hemoglobin [Hbtotal] (λ3 = 0.57 cm, ε2 = 11.0 cm-1 mM heme-1): 

[ℎ ] =  ∗ 

 

 (Eq. 2)

The percent oxidation of each sample presented in Table 1 was calculated by dividing the concentration of oxidized hemoglobin [Hb:Fe3+] by the total hemoglobin concentration [Hbtotal]. The total Hb concentrations were then multiplied by the volume of each batch to obtain molar yields (mM heme). Mass yields were calculated by dividing molar concentrations by the number of hemes per Hb (HbA = 4, LtEc = 144, GdHb = 1) and multiplying by the corresponding molecular weights (HbA = 64 kDa [36], LtEc = 3.6 MDa [30], GdHb = 16kDa [37]). Oxygen Affinity (P50) and the Hill Coefficient (n) Oxygen equilibrium curves for each sample were measured with a Hemox Analyzer (TCS Scientific, New Hope, PA). The sample was exposed to air until the partial pressure of O2 (pO2) reached 150 mm Hg of oxygen, then the sample was sparged with pure nitrogen until the pO2 decreased to 2 mm Hg. The P50 value was calculated as the pO2 at which half of the hemoglobin was bound to O2 (Y = 50%). The Hill coefficient (n) was calculated using the formula: log #$

!"

!"

&"

 = % log '   (Eq. 3) ()

where n is the Hill coefficient, [O2] is the unbound oxygen concentration, P50 is the oxygen concentration at half saturation (Y = 50%), and HbO2 is the fraction of occupied O2 binding sites. A Hill coefficient greater than one represents positive cooperativity and suggests allosteric effects between subunits, while n = 1 suggests non-cooperative binding. Quantification of Hemoglobin Concentration/Yield The (heme) concentration of each sample was determined by diluting it until its absorbance at 540 nm was between 0.1 and 1.0. The following equation was then used to calculate the corresponding heme concentration: * (- ℎ) =

(/) ∗0 ∗

(Eq. 4)

In this equation, C is the concentration of heme in mM, A540 is the absorbance at 540 nm, DF is the dilution factor, ε is the extinction coefficient (13.80 cm-1 mM-1), and l is the path length. This equation was used to calculate the concentrations of each sample at different steps in the process. Statistical Analysis All statistical analyses were performed using R Studio software (Boston, MA). Statistical significance was defined as p < 0.05. All analyses were either parametric analysis of variance (ANOVA) and Tukey’s Honestly Significant Difference following tests of normality and homogeneity of variances, or nonparametric Kruskal-Wallis analysis of variance and Post Hoc analysis if either normality or homogeneity of variances assumptions were not met. Data that initially do not meet the normality or homogeneity of variances assumptions can re-analyzed by a parametric analysis through a log transformation. A log transformation of a data set involves taking the logarithm of every data point in the data set. Once the transformation is applied, the data are tested again for normality and homogeneity of variances. If both tests pass, the transformed data are analyzed via parametric analysis. When original data and transformed data fail to meet the normality and homogeneity of variances assumptions then the original data is analyzed via nonparametric analysis. Results Metal Screening Previous studies have already shown that HbA can be effectively precipitated with Zn2+ [25,26]. Our results (see Figure 1, images of these experiments are also provided in the supplementary material) reveal that LtEc can also be completely precipitated by Zn2+ at a range of different concentrations (1.25 mM – 500 mM with 500 µM heme). This Zn2+ precipitation phenomenon motivated us to determine whether other metal ions (Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Cu2+, Cd2+, Pb2+, Fe2+, Fe3+, V3+, and Cr3+) could also precipitate HbA and LtEc. Out of all these metals, only Fe3+, Cu2+, Pb2+, and Cd2+ were able to precipitate HbA. However, precipitation only occurred at the highest concentration tested (500 mM) and significant oxidation was observed with each of these metals. In contrast, completely different precipitation and oxidation trends were seen with LtEc. In addition to Zn2+, multiple metal ions also completely precipitated LtEc above a minimum concentration of 0.5 mM, including Cu2+, Cd2+, and Pb2+. It is interesting to note that these are the same metal ions that precipitated HbA at only the highest concentration tested (500 mM). Additional metals also precipitated LtEc (Cr3+, Co2+, and Ni2+), but within a specific window of concentrations. For example, Ni2+ precipitated LtEc at concentrations between 5-125 mM, but no precipitation was observed at 1.25 or 500 mM. Each of the remaining metals (Mg2+, Ca2+, V3+, Mn2+, Fe2+, and Fe3+) provided some partial precipitation of LtEc at various concentrations, but none of these metals were able to completely precipitate LtEc. Much like HbA, significant

oxidation of LtEc was observed with Cu2+, Fe2+, Fe3+, Cr3+, and V3+, but no oxidation of LtEc was observed with Zn2+, Ni2+, Co2+, or Cd2+. Since Zn2+ completely precipitated both HbA and LtEc without any noticeable oxidation, it was selected for full scale purification experiments. Ni2+ was also chosen for LtEc purification, since it also completely precipitated LtEc (at 5-125 mM) without noticeable oxidation. Cr2+ and Cu2+ also precipitated LtEc, but were eliminated due to relatively high oxidation levels (i.e., brown pellets were observed in these samples). Cd2+ and Pb2+ precipitated LtEc without oxidation, but were not pursued due to their potential toxicity. Purification of HbA and LtEc by Zn2+ and Ni2+ PAGE analysis of samples throughout the purification process is shown in Figure 3. In the case of HbA, the crude sample exhibits strong bands at ~64 kDa, ~32 kDa, and ~16 kDa which correspond to the HbA tetramer, dimer, and monomer, respectively. HbA monomer subunits have MW’s of ~15 kDa and ~16 kDa for alpha and beta, respectively (PDB ID’s: P69905 and P68871). It is important to note that this crude HbA sample also contains many other impurities, but they are present at relatively low concentrations. However, precipitation of the HbA by Zn2+ produces a clear supernatant that can be concentrated 1,000-10,000 fold to reveal these impurities (see Lane 5 of the HbA-Zn gel in Figure 3). A dark band at approximately 32 kDa is also observed in this lane, which probably represents a very small amount of HbA (dimer) that was not precipitated, since this concentrated sample had a slight red hue. After resuspension of the precipitated HbA with EDTA and subsequent 10 kDa TFF purification, the sample appears to be highly pure, since only the bands corresponding to HbA monomers, dimers, and tetramers are observed. However, an additional unidentified band near 25 kDa is also observed in all of the HbA samples. This band has been previously observed in other HbA purification studies and may correspond to an HbA degradation/aggregation artifact generated during PAGE sample preparation [26]. Nonetheless, Lane 3 of the gel shows that Zn2+ precipitation is able to remove several impurities from the HbA sample. Regarding LtEc, the initial crude sample contains many more impurities than crude HbA, since LtEc is an extracellular protein. However, Zn2+ and Ni2+ precipitation are still able to remove most of these impurities (see Lane 5). Many of these impurities are unidentified, but one of the darker bands around 50-80 kDa may correspond to the catalase of Lumbricus terrestris (MW ~ 57 kDa) [38]. Both of the resuspended LtEc samples in Lane 7 appear to be highly pure, since only a few major bands are observed which correspond to the globin (MW = 16-17.5 kDa; PDB ID’s: P13579.1, P02218.2, P11069.3, P08924.1, 1X9F_D) and linker subunits (MW = 2532 kDa; PDB ID’s: 2GTL_M, AAF99389, 2GTL_N, ABB71122.1, 2GTL_O, ABB71123.1, ABB71124.1). The additional bands at 32, 48, or other higher molecular weights may correspond to globin dimers or trimers. Compared to samples purified by TFF alone, the samples purified by both metal precipitation and TFF appear to have similar purities (see Lanes 9 and 10 of each gel). An unidentified impurity band is observed in the purified samples around

40 kDa, but it is clear that Zn2+ and Ni2+ can be used to remove a large number of impurities from the LtEc samples. Purification of GdHb by Zn2+ Since metal precipitation was able to effectively purify both HbA and LtEc, this method was tested on an additional hemoglobin from the bloodworm Glycera dibranchiata. The hemoglobin of G. dibranchiata (GdHb) can exist as either a myoglobin-like monomer (MW = 16-17 kDa) or a polymer (MW = ~125 kDa) [32,39,40]. GdHb is also an intracellular hemoglobin like HbA, but it is expressed within nucleated red blood cells that may contain many more impurities than human red blood cells, making GdHb an interesting target for purification. Precipitation of GdHb was attempted with both 100 mM Zn2+ and Ni2+. Like HbA, precipitation of GdHb was only observed with Zn2+ and no precipitates were observed after addition of Ni2+. However, in contrast to HbA and LtEc, addition of Zn2+ to the crude GdHb sample only provided partial precipitation; both a red pellet and a red supernatant were obtained at a Zn2+ concentration of 100 mM. To determine why the GdHb did not completely precipitate, samples of the red precipitate and red supernatant were run on denaturing and native PAGE gels (see Figures 2 and 3). The denaturing/reducing gel clearly shows that each sample – crude GdRBC lysate, the red supernatant, and the red precipitate – produces a single band near 11 kDa that corresponds to the GdHb monomer. However, some key differences between the samples are revealed in the native gel. The crude GdHb lane clearly shows two bands that represent the monomeric (bottom) and polymeric (top) GdHb fractions. In contrast, the lane for the red supernatant obtained after Zn2+ precipitation only shows an upper band, suggesting that it primarily contains polymeric GdHb. The precipitated sample, however, shows a double band pattern that is similar to the crude GdHb sample, suggesting that it contains monomeric and polymeric GdHb. Altogether, these results imply that precipitation of the crude GdHb sample by Zn2+ may fractionate the GdHb into a supernatant that contains mainly polymeric GdHb and a pellet that contains monomeric and polymeric GdHb. The single monomeric band observed in the denaturing/reducing gel for the red supernatant containing polymeric GdHb suggests that the GdHb polymer may be held together by disulfide bonds that can be broken with a reducing agent (β-mercaptoethanol). These results agree with a previous study by Nagel et al, which showed that monomeric GdHb could be dimerized by adding oxidizing agents (Cu*(o-phenanthroline)2) to create disulfide bonds [32]. Since it is possible that this polymerization may block the binding site for Zn2+ and prevent precipitation of polymeric GdHb, precipitation of crude GdHb was attempted in the presence of the reducing agent dithiothreitol (DTT, 0.01-50 mg/mL). DTT may break the intermolecular disulfide bonds and dissociate the polymeric GdHb into a monomeric GdHb that is precipitated by Zn2+. Indeed, addition of DTT to the crude GdHb sample increased the size of the precipitated GdHb pellet. However, complete precipitation of GdHb was not achieved, even at

higher concentrations of Zn2+ (see sample images in supplementary material). Nonetheless, the GdHb pellet that did precipitate was purified and the supernatant fraction was discarded. Oxidation and Yield of the Purified Hemoglobins Table 1 shows the heme oxidation level of each sample immediately after purification, either by a combination of metal precipitation and TFF (e.g., HbA-Zn + TFF) or TFF alone (e.g., HbA-TFF). There is no significant difference between HbA-Zn + TFF and HbA-TFF. There is also no significant difference between LtEc-Zn + TFF and LtEc-TFF, LtEc-Ni + TFF and LtEcTFF, or between LtEc-Zn + TFF and LtEc-Ni + TFF. However, it is interesting to note that the GdHb sample does have a significantly higher oxidation level than the HbA sample. Table 1 - Final Oxidation Results Hemoglobin Metal Oxidation% HbA-Zn + TFF Zn 2.4 ± 1.3% HbA-TFF N/A 4.3 ± 3.0% LtEc-Zn + TFF Zn 5.3 ± 2.1% LtEc-Ni + TFF Ni 8.4 ± 1.2% LtEc-TFF N/A 6.4 ± 1.9% GdHb-Zn Zn 8.6 ± 0.2% The yields of HbA and LtEc on a heme and mass basis are shown in Table 2. The metal precipitation purification of HbA yielded on average 1.12 ± 0.61 grams of hemoglobin per 50 mL red blood cells. The metal precipitation purification of LtEc yielded on average 9.51 ± 1.22 grams of erythrocruorin and 3.25 ± 1.20 grams of erythrocruorin for zinc and nickel, respectively. The yield of LtEc using zinc precipitation was significantly higher than the yield of LtEc using nickel precipitation. Table 2 – Metal Precipitation Yield Data Hemoglobin Metal µmol Heme Grams Hb HbA-Zn Zn 6.67 ± 3.80 1.12 ± 0.61 LtEc-Zn Zn 3.80 ± 0.49 9.51 ± 1.22 LtEc-Ni Ni 1.30 ± 0.48 3.25 ± 1.20 Oxygen Affinity and Cooperativity of the Purified Hemoglobins The oxygen affinity (represented by P50) and cooperativity (represented by the Hill Coefficient, n) of the Hb samples purified by metal precipitation and TFF alone were measured at 25oC to determine if metal precipitation and subsequent resuspension by the chelating agent EDTA had any effect on the oxygen transport properties of each Hb (Figure 4). The effects of EDTA on LtEc may be especially important, since Ca2+ has previously been shown to influence the P50 of LtEc [41]. However, Figure 4 shows that there is no significant difference between the oxygen affinities of crude LtEc (P50 = 18.74 mm Hg), TFF-purified LtEc (P50 = 18.19 mm Hg), and LtEc purified by Zn2+ or Ni2+ precipitation (P50 = 18.18 and 18.19 mm Hg, respectively).

The HbA samples also showed no significant differences in oxygen affinity amongst themselves (P50 = 3.34-3.66 mm Hb), but they were almost all significantly lower than the LtEc samples according to nonparametric analysis. This dramatic decrease in P50 (increase in oxygen affinity) is due to removal of 2,3-DPG (an allosteric effector for HbA) during TFF and metal precipitation [27,42]. Regarding cooperativity, no significant differences were observed between the purified and crude HbA samples (n = 2.20-2.59) or between the HbA and LtEc samples. However, it is interesting to note that there was no difference in the cooperativity of the purified LtEc samples (LtEc-TFF = 2.29, LtEc-Zn = 2.37, and LtEc-Ni = 2.30), but these values were significantly lower than the cooperativity of the crude LtEc sample (n = 2.77). Since excess Zn2+ has been previously shown to increase the oxygen affinity (i.e., decrease the P50) of HbA [27], P50 and n values of each Hb were also compared to Hb’s with 0.5 mM Zn2+ and Ni2+ in the case of LtEc. Both HbA and LtEc were soluble upon addition of metal at 0.5 mM. While 0.5 mM metal did precipitate 50 µM HbA and LtEc, the samples being tested in the Hemox cuvette were much more dilute. Indeed, Figure 4 shows that addition of Zn2+ to both HbA (HbA + 0.5 mM Zn = 3.98 mm Hg) and LtEc (LtEc + 0.5 mM Zn = 23.80 mm Hg) significantly increased P50 values relative to the metal purified samples (HbA-Zn = 3.34 mm Hg, LtEc-Zn = 18.18 mm Hg). A similarly significant increase in P50 was caused by addition of 0.5 mM Ni2+ to purified LtEc (LtEc + 0.5mM Ni = 25.14 mm Hg, LtEc-Ni = 18.19 mm Hg). Both metals also significantly decrease the cooperativity of LtEc and HbA. These results show that Zn2+ and Ni2+ significantly increase the oxygen affinity and reduce the cooperativity of LtEc. In addition, these significant differences between the samples, with and without metal added, demonstrate that all of the excess metal added to the Hb’s during precipitation is effectively removed by TFF. Otherwise the samples would have similar P50 values and the P50 of the metal purified samples would be significantly higher than the samples purified by TFF alone. In the case of GdHb, the oxygen transport properties of freshly isolated G. dibranchiata red blood cells (GdRBC’s) were compared to the GdHb fraction that precipitated in the presence of Zn2+ (GdHb-Zn) and the GdHb fraction that did not precipitate (GdHb Supernatant). No significant differences in P50 were observed between the GdRBC’s and the precipitated GdHb or non-precipitated GdHb samples but there was a significant difference between all three compared to samples with additional added zinc (GdHb + 0.5 mM Zn). Cooperativity values of GdHb + 0.5 mM Zn, GdHb-Zn, and GdRBC’s weren’t significantly different. Cooperativity values of GdRBC’s and GdHb supernatant weren’t significantly different. However, the supernatant fraction did have significantly different cooperativity values than samples with excess zinc (GdHb + 0.5 mM Zn) and the resuspended pellet (GdHb-Zn). The cooperativity of the GdHb samples (n = 1.27-1.59) were significantly lower than almost all of the LtEc and HbA samples (n = 2.2-2.8) analyzed via nonparametric analysis. However, low cooperativity is expected for a monomeric hemoglobin like GdHb and the monomeric fraction of GdHb has been observed to be non-cooperative [43]. Finally, it is also interesting to note that addition of 0.5 mM Zn2+ significantly increased the P50 of GdHb (GdHb + 0.5 mM Zn = 17.15 mm Hg) relative to all of the other GdHb and GdRBC samples (P50 = 12.19-14.17 mm Hg), just as it did for HbA and LtEc. Therefore, divalent cations like Zn2+ and Ni2+ seem to significantly reduce the oxygen affinity (and increase the P50) of a wide variety of structurally different Hb’s, including HbA, LtEc, and GdHb.

Discussion It has been previously shown that HbA can bind Zn2+ ions in its 2,3-diphosphoglycerate binding site [27] and that LtEc binds several different metals on its surface (e.g., Ca2+, Cu2+, & Zn2+) [44]. It is possible that LtEc binds to many more metals that HbA and GdHb, since its surface is covered in hundreds of metal-binding amino acids such as histidine, glutamic acid, and aspartic acid (PDB ID: 2GTL). Our results (Figure 1) reveal that LtEc does bind several different metal ions, including the toxic heavy metals Cd2+ and Pb2+. These findings may be of particular relevance to previous bio-accumulation studies, which show that earthworms consume and accumulate many different heavy metals (e.g., Cd2+) from contaminated soils [45–47]. It has been shown that earthworms and other invertebrates are able to tolerate exposure to these heavy metals by using the ample supply erythrocruorin in their bloodstreams to bind and sequester the toxic metal ions [48]. Future work involving LtEc extraction and characterization from earthworms exposed to contaminated soils will be necessary to confirm this hypothesis. Although it was observed that LtEc could be precipitated by many different metal ions (see Figure 1), Zn2+ and Ni2+ were selected as leads because they completely precipitated LtEc without any noticeable oxidation of the heme iron (as seen with Cu2+ or Fe3+) or potential toxicity (as known for Cd2+ or Pb2+). These metals were used to develop a purification technique in which Hb’s are selectively precipitated by metal ions, then resuspended by adding a chelating agent (EDTA) to sequester the metal ions. The resuspended Hb samples are sterilized with a 0.2 µm TFF filter and then excess metal/EDTA complexes and other impurities are removed with a 500 kDa (for LtEc) or 10 kDa (for HbA and GdHb) TFF filter. This method produces highly pure Hb samples with a high yields and low heme oxidation levels (see Figure 3 and Tables 1-2). Most importantly, the oxygen transport properties of each Hb are not affected by metal precipitation or resuspension by EDTA. These results show that metal (Zn2+) precipitation coupled with tangential flow filtration is a particularly effective strategy for purifying a wide array of hemoglobins with vastly different structures, including HbA and LtEc. This method was also used to successfully purify other erythrocruorins, including Nephelopsis obscura, Alitta virens, and Eisenia fetida (data not shown). However, GdHb was not completely isolated using Zn2+ precipitation. It is possible to precipitate almost all of the GdHb by adding a reducing agent (DTT) prior to Zn2+ precipitation, but a slightly red supernatant is still observed. These results suggest that GdHb has a highly specific Zn2+ binding site with a cysteine residue that may (1) be required for Zn2+ binding or (2) may sterically hinder Zn2+ binding via disulfide bond formation and GdHb polymerization. Finally, one additional benefit of metal precipitation is that it removes a large amount of impurities and aggregates from crude LtEc samples that may clog the 0.2 µm TFF membrane used for sterilization. Indeed, while it was previously observed significant fouling of the 0.2 µm membrane by these impurities without metal precipitation, membrane fouling with metal precipitation was not observed [18]. Therefore, the metal precipitation step may simultaneously increase product purity and prolong the life of TFF filters used for LtEc purification. Conclusions

Donated blood has always been the ideal material for a blood transfusion, but its limited shelf life and availability has made the search for viable hemoglobin-based oxygen carriers (HBOC’s) necessary. Due to the demand that would be required by large scale HBOC production, a suitable and easily scalable purification method is needed. This need has been addressed by developing a purification strategy that uses Zn2+ to selectively precipitate Hb’s, followed by desalting and removal of additional impurities with tangential flow filtration. This method provides high yields of Hb products with purity, oxidation levels, and oxygen transport properties that are identical to Hb’s purified by TFF alone. Therefore, zinc precipitation is a simple and easily scalable method that provides large quantities of functional hemoglobin for the development of blood substitutes. Acknowledgements The authors would like to thank the Villanova Undergraduate Research Fellows program for financially supporting Jack Dienes, as well as Drs. Daniel Kraut and Michael Russell of Villanova University for assistance in PAGE troubleshooting and statistical analysis, respectively. References [1]

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Figure Captions Scheme 1. Hemoglobin Purification Process. Metal was added to crude Hb/Ec samples (1) to precipitate the Hb/Ec, leaving a clear supernatant (2). The supernatant was decanted and 100 mM EDTA was added to resuspend the pellet (3). The resuspended Hb/Ec was then filtered through a 0.2 µm tangential flow filter (TFF). The 0.2 µm filtrate was then further purified via 10 rounds of diafiltration on a second TFF filter (500 kDa molecular weight cut off, MWCO, for LtEc, 10 kDa MWCO for HbA and GdHb) to provide highly pure Hb/Ec (4). Figure 1 – Hemoglobin Metal Precipitation Screen. An asterisk (*) indicates that oxidation was observed with the corresponding metal. The dashed lines indicate the range of concentrations in which each metal was able to precipitate HbA or LtEc (e.g., Cr3+ precipitated LtEc at concentrations of 1.25-50 mM). Figure 2 –PAGE Analysis. Samples were run on 10% tricine gels in the following order: Lane 1 – protein molecular weight ladder, Lane 3 – crude sample, Lane 5 – impurities in supernatant after metal precipitation of Hb’s, Lane 7 – resuspended Hb/Ec pellets, Lane 9 – Hb/Ec samples after TFF purification, and Lane 10 – Ec samples purified with only TFF. Figure 3 –Native Gel Analysis. All purified samples were run on a native protein gel to compare polymerization of GdHb fractions. Figure 4. Hemoglobin Oxygen Affinity Analysis. Representative oxygen equilibrium curves (OEC’s) are shown in A, D, and G for HbA, LtEc and GdHb, respectively. P50 values were obtained from OEC data. Cooperativity (Hill Coefficient or n) values were calculated using Eq. 3. HbA and LtEc P50 and cooperativity data (B, E and C, F) were analyzed via a parametric analysis. LtEc P50 data were log transformed (see Statistical Analysis). GdHb P50 data (H) were analyzed via a parametric analysis. GdHb cooperativity data (I) were analyzed via a nonparametric analysis. All samples were analyzed in Tris buffer at 25 °C. Number of replicates are: HbA Crude (n=6), HbA-Zn (n=9), HbA-TFF (n=3), HbA + 0.5 mM Zn (n=3), LtEc Crude (n=7), LtEc-Zn (n=7), LtEc-Ni (n=7), LtEc-TFF (n=3), LtEc + 0.5 mM Zn (n=3), LtEc + 0.5 mM Ni (n=3), GdRBC’s (n=3), GdHb-Zn (n=8), GdHb Supernatant (n=3 for P50, n=2 for cooperativity), and GdHb + 0.5 mM Zn (n=3). Vertical error bars represent 90th percentile (upper) and 10th percentile (lower). Boxes represent 75th percentile (upper boundary), 50th percentile (center line), and 25th percentile (lower boundary). Dots represent 95th percentile (upper dot) and 5th percentile (lower dot). Means ± standard deviation are reported below sample name. Samples not statistically different are grouped under the same line.

Scheme 1

Figure 1

Figure 2

Figure 3

Figure 4

Highlights • • • •

Zn2+ and Ni2+ can be used to selectively precipitate L. terrestris erythrocruorin (LtEc) Only Zn2+ ions selectively precipitate human (HbA) and G. dibranchiata (GdHb) hemoglobin GdHb is present as monomers and polymers and Zn2+ may allow for selective purification of different fractions Coupling metal precipitation with tangential flow filtration produces a highly pure hemoglobin/erythrocruorin product with minimal oxidation