Direct comparison of oligochaete erythrocruorins as potential blood substitutes

Abstract While many blood substitutes are based on mammalian hemoglobins (e.g., human hemoglobin, HbA), the naturally extracellular hemoglobins of invertebrates (a.k.a. erythrocruorins, Ecs) are intriguing alternative oxygen carriers. Specifically, the erythrocruorin of Lumbricus terrestris has been shown to effectively deliver oxygen in mice and rats without the negative side effects observed with HbA. In this study, the properties of six oligochaete Ecs (Lumbricus terrestris, Eisenia hortensis, Eisenia fetida, Eisenia veneta, Eudrilus eugeniae, and Amynthas gracilis) were compared in vitro to identify the most promising blood substitute candidate(s). Several metrics were used to compare the Ecs, including their oxidation rates, dissociation at physiological pH, thermal stability, and oxygen transport characteristics. Overall, the Ecs of Lumbricus terrestris (LtEc) and Eisenia fetida (EfEc) were identified as promising candidates, since they demonstrated high thermal and oligomeric stability, while also exhibiting relatively low oxidation rates. Interestingly, the O2 affinity of LtEc (P 50 = 26.25 mmHg at 37 °C) was also observed to be uniquely lower than EfEc and all of the other Ecs (P 50 = 9.29–13.62 mmHg). Subsequent alignment of the primary sequences of LtEc and EfEc revealed several significant amino acid substitutions within the D subunit interfaces that may be responsible for this significant change in O2 affinity. Nonetheless, these results show that LtEc and EfEc are promising potential blood substitutes that are resistant to oxidation and denaturation, but additional experiments will need to be conducted to determine their safety, efficacy, and the effects of their disparate oxygen affinities in vivo.

). 2,9,10 New HBOC products are being developed to solve these problems, including Oxy-Vita (a higher MW "zero-link" polymerized bHb), [11][12][13][14] HemoTech (an anti-inflammatory ATP cross-linked bHb), 15 and pPolyHb (a polymerized porcine Hb), but clinical data are not yet available for these products. 16,17 The limited success of intracellular mammalian Hbs has also motivated researchers to investigate the naturally extracellular hemoglobins of invertebrates (aka Erythrocruorins or Ecs). Ecs from a variety of organisms have been studied, including annelids, 18 mollusks, 19 insects, 20,21 snails, 22,23 and many more. 24 Overall, the most thoroughly investigated Ecs are from the annelids Lumbricus terrestris and Arenicola marina, which are both huge macromolecular complexes (MW $3.6 MDa). For example, the structure of LtEc (obtained via X-ray crystallography) consists of 144 globins and 36 linker subunits. 25 LtEc assembly begins when 4 globins form a tetramer, which can then associate with other tetramers to form a dodecamer ($208 kDa). The linker chains bind the dodecamers to form a protomer that associates with 11 other protomers to yield a hexagonal bilayer (HBL) of protomers that is $30 nm across.
The Ec of the marine polychaete A. marina (AmEc, also known as Hemarina®) has been successfully utilized for oxygen preservation in tissue culture, 26 oxygen transfer in bioreactors, 27 and organ preservation. 28 AmEc has also been successfully transfused into mice and hamsters without eliciting an immune response or significant changes in blood pressure. 29,30 However, AmEc was observed to quickly dissociate from the HBL into dodecamers when exposed to the relatively low ionic strength of human plasma in in vitro studies conducted at 378C, pH 7.4. 29 In contrast, the Ec of the terrestrial oligochaete L. terrestris (LtEc) does not dissociate in human plasma. 31 However, transfusions of both AmEc and LtEc effectively maintain oxygen delivery in mice, rats, and hamsters without eliciting an immune response or increase in blood pressure. [31][32][33] Altogether, these promising results indicate that both AmEc and LtEc could be safe and effective blood substitutes. 25 In addition to AmEc and LtEc, several other Ecs with unique properties have also been described. 24,34 For example, the oxygen affinity of some Ecs can be relatively high, including the polychaete Branchipolynoe symmytilida (P 50 5 0.9-1.4 mmHg at 208C, pH 7.5) 35 and the giant ($3 m in length 36 ) Gippsland worm (P 50 5 2 mmHg at 258C, pH 7.5). 37 In contrast, the "chlorocruorins" of Eudistylia vancouverii and Potamilla leptochaeta have a modified heme group that gives them a green appearance and significantly decreases their oxygen affinity (P 50 5 145 mmHg and 155 mmHg, respectively, at 208C, pH 7.4). 38,39 Meanwhile, the Ecs from Riftia pachyptila and Oligobranchia mashikoi posess a unique spherical structure that consists solely of globin subunits (MW $400 kDa). 40,41 Annelid Ecs have also been discovered in extreme environments. For example, the marine worm Alvinella pompejana is a hydrothermal vent dwelling marine worm that thrives in an environment that is anoxic, rich in CO 2 and sulfide, and can tolerate temperatures that vary from 2 to 3508C. 42,43 While the unique properties of these exotic Ecs may be attractive, many of these species are prohibitively rare or difficult to obtain. In contrast, terrestrial oligochaete worms are available in relatively large quantities and low costs due to their prevalence in the bait and composting industries. Many of these terrestrial worms have been studied individually (e.g., LtEc 25 and Glossoscolex paulistus (GpEc) [44][45][46][47][48] ), but very few direct comparisons of their properties are available. 34,45 The purpose of this study is to directly compare the biophysical properties of six erythrocruorins from commercially available oligochaetes, including Lumbricus terrestris (LtEc, Canadian nightcrawler), Eisenia hortensis (EhEc, European nightcrawler), 49 Eisenia fetida (EfEc, red wiggler), 50 dissociated during purification, possibly due to the oxidation of the heme iron, thereby allowing it to permeate the 500 kDa filter. Nonetheless, the relatively small amount of red retentate sample obtained for AgEc was used for subsequent experiments. The oxidation level of the purified AgEc sample was relatively high (24.7%, see Table 1) but the oxidation levels of the other purified Ecs (shown in Table 1) were all relatively lower (4-15% Fe 31 ).

| PAGE analysis
Following TFF purification, all Ecs were analyzed on a 10% acrylamide/ glycine gel (see Figure 1). HbA and bHb were also included as MW standards that contain only globin subunits (16 kDa; no linker subunits).
Overall, each Ec appeared to be highly pure, showing only the characteristic Ec band pattern with multiple globin monomers around 15-18 kDa and multiple linker subunits ranging from 24 to 32 kDa. However, while EvEc, EfEc, LtEc, and EhEc all displayed at least three distinct globin bands, AgEc and EeEc only appeared to have two distinct globin bands.
This observation suggests that the MW of one of the AgEc and EeEc globins may be significantly different than the other Ecs. It is worth noting that the A subunit of LtEc was reported to be glycosylated with glycans that are 1.4-1.9 kDa. 55 Therefore, this difference in band patterns may reflect a difference in glycosylation of one of the subunits, but without primary sequence data for AgEc and EeEc it is unclear why they lack a higher MW globin band around 18 kDa. Nonetheless, the band patterns of the linker subunits of each Ec were highly similar. EvEc, EfEc, AgEc, and EhEc did display higher MW bands, but these may be attributed to unreduced disulfide-linked ABC trimers (MW $48 kDa), which are a common structural feature of Ecs. 56 Future experiments with electron spray ionization mass spectrometry will need to be conducted to determine the exact MW of each Ec and their glycosylation patterns.

| Size exclusion chromatography: Structural stability
The SEC elution profiles of each purified Ec are shown in Figure 2 (mobile phase 5 20 mM Tris, pH 7.4). The elution profile of HbA is also shown as a MW standard (MW HbA 5 64 kDa), along with the hemoglobin of the bloodworm Glycera dibranchiata (GdHb), which includes a monomeric fraction (MW GdHb monomer 5 16 kDa) 57 and a polymeric fraction (MW GdHb polymer 5 108 kDa). 58 As expected for high MW proteins (e.g., MW LtEc 5 3.6 MDa), each Ec exhibited at least one high MW fraction (detected via absorbance at 280 nm) that quickly eluted from the column after 19 min. However, a second red fraction also eluted 4 min later for the EvEc, EhEc, and EeEc samples. The absorbance spectrum of the second red fraction observed with EvEc, EhEc, and EeEc was similar to most hemoglobins (data not shown), suggesting that globin subunits were present in the sample. The EeEc sample also displayed a minor third fraction with an elution time similar to HbA tetramer (28.5 min), but it was colorless. PAGE analysis revealed no change in band patterns between the SEC-separated peaks and the original sample, indicating the presence of linker proteins. Therefore, it appears that EvEc, EeEc, and EhEc may have dissociated during tangential flow filtration (as was visibly observed during purification of AgEc) or it could reflect a structural instability of these Ecs at pH 7.4. Indeed, many other Ecs have been shown to dissociate at alkaline pH. 59-61

| Thermal stability
The thermal stability of the Ecs was compared by measuring their melting temperatures (T m ) with a thermal shift assay ( Figure 3). In this assay, SYPRO Orange dye binds to hydrophobic residues that are exposed as proteins denature at higher temperatures, causing an increase in dye fluorescence that can be quantitatively measured. The T m is then  62 and HbA (T m 5 558C, data not shown).
Since EvEc, EhEc, and EeEc separated into two distinct fractions during SEC, the melting temperatures of those individual peaks were also compared ( Figure 3, right). In each case, there were no significant differences in T m between the two fractions of each Ec. However, it is interesting to note that both of the SEC-purified EeEc fractions (55-568C) were significantly more thermally stable than the TFF-purified EeEc (518C). These results suggest that a pro-oxidant impurity (e.g., low MW protein or metal ion) may have been present in the TFF-purified EeEc sample and then removed during SEC.

| Oxygen affinity and cooperativity
Oxygen equilibrium curves for each Ec at 378C in Hemox buffer are displayed in Figure 4, while their calculated oxygen affinity (P 50 ) and

| Oxidation rate analysis
The oxidation of each Ec (Fe 21 to Fe 31 ) in Tris buffer (pH 7.4) and Ringer's Lactate solution (pH 7.4) at 258C is shown in Figure 5, while the calculated oxidation rates (k ox ) are shown in Table 3. In Tris buffer, all of the Ecs oxidized much faster than HbA (k ox 5 0.55 x 10 23 hr 21 ).
Interestingly, the two highest oxidation rates were observed with EhEc and EvEc (3.4 x 10 23 and 9.8 x 10 23 hr 21 , respectively), which were also observed to dissociate at pH 7.4 (see Figure 2). Other studies have shown a similar phenomenon, in which dissociation of Ecs significantly increases their oxidation rates. 64,65 In contrast, all of the trends observed in Tris buffer were reversed  have been shown to decrease the oxygen affinity of HbA (D94H, P124Q, and G136D, respectively). [72][73][74] Overall, any or all of these isolated mutations could be responsible for the relatively low oxygen affinity of LtEc, but future mutational studies would be needed to confirm this hypothesis. The circulation half-life of both LtEc and AmEc are limited to 12 hours, 33,75 but PEGylation of LtEc has been shown to increase its halflife up to 70 hours. 76 Transfusions of LtEc and AmEc also do not elicit any changes in animal behavior or health for several months after the initial injection. 29,31 In addition, hyper-responsive BP/2 mice injected with AmEc do not produce significant antibody titers against AmEc. 29 All of these preliminary results are promising, but further work must be conducted to characterize the potential immunological effects of other Ecs and determine the potential need for other functions besides oxygen transport (e.g., carbon dioxide transport). 77  fied with ten successive rounds of diafiltration on a 10 kDa TFF filter as previously described. 78

| Preparation of crude Ecs
Approximately 500-1,000 worms of each species were purchased from various suppliers. Lumbricus terrestris specimens were purchased Ec sample by 10 successive rounds of diafiltration using a 500 kDa TFF filter as previously described. 33 Specifically, in each round of diafiltration, the red retentate was concentrated 10-fold and then diluted again (e.g., 2500 to 50 ml) with 20 mM Tris buffer, pH 7.0 at 48C. After the final concentration step, the cyanmethemoglobin assay 79 was performed to measure oxidation levels and the samples were frozen at 2728C until needed. heme :

| Cyanmethemoglobin oxidation assay
For each Ec, a separate sample was diluted by a factor, D 2 , to a total The percent oxidation of each hemoglobin sample, shown in Table   1

| SEC analysis
Each Ec and Hb was analyzed using a NGC TM Chromatography System  Figure 2 were normalized such that the maxima of the first elution peak were held constant.

| PAGE analysis
where n is the Hill coefficient, pO 2 is the unbound oxygen concentration and HbO 2 is the fraction of occupied O 2 binding sites. In this context, Hill coefficients greater than one represent positive cooperativity and allosteric interactions between subunits, while n 5 1 suggests noncooperative O 2 binding. The absorbance spectra of the samples were measured daily for 2 weeks and then used to estimate oxidation levels using Equation 4.

| Oxidation rate analysis
Since the plots of ln(%Fe 21 ) versus time for each Ec appeared to be linear, a single exponential decay model (Equation 5) was used to estimate oxidation rates for each Ec.

| Statistical analysis
All statistical analyses were performed using R Studio software (Boston, MA) or by simultaneous T tests in Microsoft Excel. Statistical significance was defined as p < .05. All analyses conducted in R Studio were parametric analysis of variance and Tukey's Honestly Significant Difference following tests of normality and homogeneity of variances.

| C O NC LU S I O N S
Altogether, these results identify LtEc and EfEc as two promising potential blood substitutes that warrant future study. They both resist oligomeric dissociation at pH 7.4, while also exhibiting relatively low oxidation rates and high melting temperatures. It is also interesting to note that these Ecs have vastly different oxygen affinities, but the implications of that difference will need to be determined in future animal studies. Finally, it would also be interesting to directly compare the terrestrial Ecs studied in this work to other well-studied marine Ecs (e.g., AmEc).

ACKNOWLEDGMENT
The authors would like to thank Carlos Rosas for his support.