Chris E.
Cooper
*a,
Gary G. A.
Silkstone
a,
Michelle
Simons
a,
Svetlana
Gretton
a,
Badri S.
Rajagopal
a,
Victoria
Allen-Baume
a,
Natalie
Syrett
a,
Thoufieq
Shaik
a,
Gina
Popa
b,
XiaoBo
Sheng
b,
Matthew
Bird
b,
Ji-Won
Choi
b,
Riccardo
Piano
c,
Luca
Ronda
cde,
Stefano
Bettati
cdef,
Gianluca
Paredi
f,
Andrea
Mozzarelli
defg and
Brandon J.
Reeder
*a
aSchool of Life Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, UK. E-mail: ccooper@essex.ac.uk; reedb@essex.ac.uk; Tel: + 44 Tel: (0) 1206 872119
bAbzena, Babraham Research Campus, Babraham, Cambridge, CB22 3AT, UK
cDepartment of Medicine and Surgery, University of Parma, 43124 Parma, Italy
dBiopharmanet-TEC, University of Parma, 43124 Parma, Italy
eInstitute of Biophysics, CNR, 56124 Pisa, Italy
fSITEIA.Parma, University of Parma, 43124 Parma, Italy
gDepartment of Food and Drug, University of Parma, 43124 Parma, Italy
First published on 15th June 2020
In order to infuse hemoglobin into the vasculature as an oxygen therapeutic or blood substitute, it is necessary to increase the size of the molecule to enhance vascular retention. This aim can be achieved by PEGylation. However, using non-specific conjugation methods creates heterogenous mixtures and alters protein function. Site-specific PEGylation at the naturally reactive thiol on human hemoglobin (βCys93) alters hemoglobin oxygen binding affinity and increases its autooxidation rate. In order to avoid this issue, new reactive thiol residues were therefore engineered at sites distant to the heme group and the α/β dimer/dimer interface. The two mutants were βCys93Ala/αAla19Cys and βCys93Ala/βAla13Cys. Gel electrophoresis, size exclusion chromatography and mass spectrometry revealed efficient PEGylation at both αAla19Cys and βAla13Cys, with over 80% of the thiols PEGylated in the case of αAla19Cys. For both mutants there was no significant effect on the oxygen affinity or the cooperativity of oxygen binding. PEGylation at αAla19Cys had the additional benefit of decreasing the rates of autoxidation and heme release, properties that have been considered contributory factors to the adverse clinical side effects exhibited by previous hemoglobin based oxygen carriers. PEGylation at αAla19Cys may therefore be a useful component of future clinical products.
One modification is to mimic the protective effects of the red blood cell via encapsulating Hb into natural or artificial vesicles.7 Alternatively covalent cross-linking of subunits – either using chemical5 or genetic8 means – can increase the minimum molecular size of Hb preventing renal clearance. If the cross-linked protein is large enough it can also decrease unwanted extravasation of the protein into the surrounding tissue, potentially preventing the undesirable scavenging of the intercellular messenger nitric oxide.9 An attractive alternative to these methods is conjugation with large unreactive organic molecules such as poly(ethylene glycol) (PEG). PEG is particularly attractive as it has been shown to decrease immunogenicity and extend the circulatory lifespan of modified proteins10 and is used in many licensed pharmaceutical products.11 PEGylation of human Hb was used in previous HBOC products in clinical trials by Sangart (MP4, Hemospan12), APEX Pharmaceuticals (PHP13) and Baxter Healthcare (rHb2.014). PEGylation of bovine Hb is used in Sanguinate®,15 a current product being trialed by Prolong Pharmaceuticals.
Activated PEGs are able to conjugate to proteins as acylating reagents, alkylating reagents, or thiol-reactive reagents.11 Human Hb has only one reactive sulfhydryl residue per dimer that can be readily PEGylated using standard thiol reactive agents such as maleimide-PEG (MAL-PEG). Modifications at this site (βCys93) increase – albeit only slightly – the oxygen affinity of Hb.16 However, of greater concern they increase the autoxidation rate of oxyhemoglobin (oxyHb) and make Hb more liable to oxidative damage17 and heme loss.18 Modifications at this site (nitrosation, glutathionylation) in vivo have also been suggested to play an important role in a signaling function of Hb that may be important to maintain in a HBOC.19 Reactivity at βCys93 can be maintained in PEGylated Hb by protecting this residue prior to modification at other surface residues.20
Maleimide-PEG conjugation at non cysteine residues is possible via the use of reagents such as 2-iminothiolane to introduce thiol reactivity at terminal amino residues and surface lysines. Such methods of PEGylation have been used successfully in the design of HBOC products tested in animal models (and clinical trials) such as Hemospan,21 Euro-PEG-Hb22 and Sanguinate®.23 However, two issues have arisen. The first is that the final product is very heterogeneous, due to differences in the efficiency at different protein sites of the reactions both creating free thiol residues and/or the subsequent PEGylation at those residues. Heterogenous products also arise when bifunctional activated PEG reagents react at primary amines in Hb as is the case with PHP. HBOCs formed in these ways can have as few as 0 or as many as 14 PEG chains bound.24,25 A heterogeneous product makes it difficult to test and control the reactivity and side reactions of Hb fractions that may be only small components of the product, but could lead to altered reactions in vivo. In this context, it is perhaps noteworthy that preclinical and clinical data led to Biopure modifying their heterogenous cross-linked HBOC products (HS1, HS-2) to create more homogenous products for veterinary (Oxyglobin®) and clinical use (Hemopure®).26
The second problem is that non-specific PEGylation frequently includes residues key to dimer–dimer interactions at the α/β interface. Therefore, these products are frequently dimeric. Although the PEGylated dimers are still too large for rapid renal filtration, they take on some of the properties of the Hb dimer (enhanced autoxidation, high oxygen affinity, no cooperativity). Although this high affinity was marketed as a useful feature in Hemospan,12 this does restrict the ability to modify Hb functions in any new product. PEGylation under controlled conditions in the absence of oxygen favors the T state of Hb, increasing the concentration of tetrameric HBOC,27 but does not completely remove the problem.24
For these reasons it would be useful to generate a HBOC that is derived from homogenous PEGylation at a site that does not perturb the functional properties of Hb. A recombinant Hb carrying a single reactive free surface cysteine at a site remote from the dimer/tetramer interface could possess these desirable qualities. Therefore, this paper tests the efficacy and functional effects of homogenous PEGylation in human Hb at βCys93 (wild type) and two new positions generated by replacing existing alanine residues with cysteine; βAla13Cys is a site homologous to a surface cysteine naturally occurring in feline Hb and αAla19C is a completely novel site on the α subunit.
Fig. 1 Stereo view of mutation sites for PEGylation. An αAla19Cys and βAla13Cys mutation introduced reactive thiol sites for PEGylation for A12 (red) and A13 (yellow) respectively. For both A12 and A13 the βCys93 site was mutated to an Alanine residue (green). α-Subunit is in purple, β-subunit in blue and heme as white stick. PDB was 1A3N, human hemoglobin (deoxy) mutations were in silico. |
HbCO (R state Hb) was tested to limit undesirable oxidative reactivity during any subsequent PEGylation and purification process. Consistent with previous findings14 there were two reactive thiol residues per α2β2 tetramer for wild type Hb (A1) at βCys93. No reaction was observed when βCys93 was converted to alanine (A11). The creation of new sulfhydryl sites in the α-subunit (A12: βCys93Ala/αAla19Cys) or the β-subunit (A13: βCys93Ala/βAla13Cys) restored this reactivity.
It is important that any new Hb mutations introduced into a putative HBOC do not increase the oxidation of Hb, nor facilitate the release of the heme cofactor. Fig. 3 shows the stability of the recombinant proteins towards autoxidation and heme loss. A12 has lower autoxidation than native Hb. A11 and A12 have lower heme loss than native Hb, whereas A13 has greater heme loss than native Hb. However, in all cases the reactivity is only slightly different to native Hb, indicating that any post translational modifications (or lack of such modifications) in the recombinantly produced proteins exert rather small effects on reactivity in the heme pocket.
SDS-PAGE (Fig. 4) shows the dominant fraction of the recombinant Hb is consistent with α and β monomers (16 kDa). Consistent with the presence of a free sulfhydryl residue, A1, A12 and A13 are all able to react with 20 kDa MAL-PEG. As seen previously for many proteins,28,29 including Hb,21,24 PEGylated protein adducts run at a slightly higher apparent molecular weight than predicted from unPEGylated protein molecular weight markers. This discrepancy between apparent and real molecular weights in SDS-PAGE of PEGylated Hb can be shown by excising the band and using mass spectrometry.24 Under the conditions of Fig. 4, barium iodide staining showed that the unreacted PEG ran at twice the apparent molecular weight compared to a protein of similar size. So in the case of the Hb monomer (16 kDa) we would expect to observe a mono-20 kDa PEGylated Hb band at ca. 56 kDa (16 kDa for the protein portion plus an apparent 40 kDa for the PEG). The gels in Fig. 4 are therefore consistent with the major new product being Hb (16 kDa) bound to a single (20 kDa) PEG. Note in all cases one subunit will be unreactive to MAL-PEG so that a band remains at 16 kDa – the α-subunit in A1 and A13 and the β-subunit in A12. The visual inspection and the densitometric analysis of gels indicate that A11, A12 and A13 preparations contain a small band at around 30–35 kDa, consistent with an unPEGylated covalent dimer. In some samples of A12 and A13 the proportion of the band in this region increased significantly. In these cases, the excess could be decreased by treatment with dithiothreitol (DTT). However, even post DTT treatment a band remained, suggesting the possibility of a fraction of covalently bound dimer other than a disulfide bridge.
LC-MS QTOF analysis revealed molecular mass for the dominant Hb species both pre and post-DTT treatment of 15289 and 15966, consistent with α and β-subunits with uncleaved N-terminal methionine residues, as is usual for recombinant Hb (see Fig. S1† for A12 data). A minor species observed was more heterogenous, with dominant masses of 30578 and 31932, consistent with the presence of αα and ββ dimers.
Sulfhydryl reactivity and PEGylation of Hb has previously been shown to be partially dependent on the conformational state of the protein (R state or T state). The natural βCys93 site (A1) is more exposed in the R state (favored in oxyHb).30 If a new reactive cysteine was introduced on the surface, distant from parts of the protein that undergo large conformational changes, this differential effect might be absent. This was confirmed in Fig. 5, which shows MAL-PEG reactivity to mutants in R-state Hb (CO-bound) or T-state (deoxygenated). Under the latter conditions, before PEGylation, when hemoglobins were deoxygenated, ascorbate (0.2 mM) was also added as a reducing agent during the reaction. Under both conditions A12 and A13 showed more efficient conjugation than A1 with approximately 50% of the Hb subunits binding PEG (as expected for reactivity at the single external residue in the α- (A12) or β-subunit (A1, A13). Again, no reactivity was seen in the mutant only lacking βCys93 (A11).
It is desirable for PEGylation to be efficient and result in a homogenous HBOC. The extent of PEGylation and the heterogeneity of PEGylated Hb derivatives were evaluated via size exclusion chromatography (SEC) (Fig. 6 and S2†), under conditions where Hb is predominantly dimeric. Results show that PEGylated A12 is more homogenous than PEGylated A13. Moreover, A1 shows a significant amount of unPEGylated protein. The SEC analysis also revealed the presence of a fraction of tetramers for both A12 and A13. This is possibly associated with the reaction of the exposed cysteines and formation of a disulfide bridge between two dimers. However, this did not seem to unduly retard PEGylation efficiency which reveals the order A12 > A13 ≫ A1.
Fig. 6 Size exclusion chromatography pre and post PEGylation. SEC of Hb mutants pre (left panel) and post (right panel) PEGylation. For conditions see Materials and methods. For an example of protein MW standards run at the same time as A12 Hb see Fig. S2.† Comparison with SEC standards reveals Hb mutant peaks at 10.8–10.9 min comprise Hb dimer and peaks at 9.9 min tetramer. Hb PEG peaks at 7.7–7.8 min comprise PEGylated dimers. The minor peak at 6.7–6.8 is not straightforward to assign without further analysis. It could be a PEGylated tetramer, but it might also be a different high MW species, e.g. nonspecific protein aggregates or possibly di-PEGylated Hb. |
The molecular mass of the MAL-PEG adducts was determined using MALDI/TOF spectrometry (Fig. 7), under oxygenated CO-saturated (oxy) and anaerobic (deoxy) conditions for A12 (Fig. 7A) and A13 (Fig. 7B). In the region 14000–17000 m/z, A12 and A13 showed both α and β peaks, while in the region 30000–34000 three peaks, corresponding to α–α, α–β, and β–β complexes appeared. This is possibly due to artifacts generated by the denaturing conditions used for the mass spectrometry and was also observed for samples of native human HbA0. Upon PEGylation in both R and T state conditions, the A12 sample (Fig. 7A) shows an almost complete disappearance of the peak corresponding to the α chain, in agreement with derivatization of the introduced α Cys19 with 20 kDa MAL-PEG that now exhibits a broad peak around 36000 m/z. Similarly, for A13 (Fig. 7B), there is an almost complete disappearance of the peak corresponding to the β chain, in agreement with derivatization of the introduced β Cys13 with 20 kDa MAL-PEG that exhibits a broad peak around 36000 m/z. It is known that PEG-conjugated proteins exhibit a low propensity to fly and, consequently, they give low signals in mass spectrometers. However, it is clear that the dominant protein product for A12 corresponds to a β-subunit and an α-subunit with a single PEG bound. Likewise, A13 predominantly comprises an α-subunit and a β-subunit with a single PEG bound.
The kinetics of MAL-PEG reactivity was explored in A12 by varying the concentration and time of incubation (Fig. 8). As there are two reactive thiols per tetramer, 100% efficiency with no side reactions or unreacted Hb would have led to 50:50 end ratios of Hb monomer to Hb–PEG adduct. Increasing the PEG:Hb ratio during incubation (from 3:1 from 12:1) and the length of the incubation (from 1–3 h) increased the efficiency of PEGylation in A12. However, the increase was rather small – judged on a per α-subunit basis – from 66% at the lowest PEG ratio (3:1) and shortest incubation time (1 h) to 82% at the highest PEG ratio (12:1) and longest incubation time (3 h).
Cooperative oxygen binding is likely to be a useful function in a HBOC and in our mutants, unusually for PEGylated Hb, cooperative oxygen binding was maintained. A12 and A13 PEGylation had no significant effect on oxygen affinity or the Hill coefficient for cooperative binding, although A1 – consistent with previous findings17 – showed a small, but significant, increase in affinity (Table 1 and Fig. S3†).
P 50 (torr) | P 50 (torr) (after PEG) | Hill coefficient | Hill coefficient (after PEG) | |
---|---|---|---|---|
P 50 measured in mmHg. *p < 0.05 compared to pre-PEGylated value. | ||||
A1 WildType (β-C93) | 5.0 ± 0.3 | 3.8 ± 0.2* | 1.7 ± 0.2 | 1.9 ± 0.2 |
A13 β-A13C/β-C93A | 4.2 ± 0.2 | 4.6 ± 0.2 | 1.9 ± 0.3 | 2.0 ± 0.2 |
A12 α-A19C/β-C93A | 4.4 ± 0.2 | 4.1 ± 0.2 | 1.6 ± 0.2 | 1.6 ± 0.1 |
As A12 showed the most efficient PEGylation of our mutants, it was characterized in more detail. PEGylation of A12 resulted in a more stable protein with a decrease in autoxidation and heme loss compared to A1 or unPEGylated A12 (Table 2, Fig. S4 and S5†). Antioxidant reductants such as ascorbate can reduce reactive damaging oxidative Hb species, such as ferryl heme. Accessibility of heme to plasma reductants, such as ascorbate, is therefore desirable to maintain in an HBOC and we tested whether the PEGylation of A12 prevented access of ascorbate to the heme. In wild type Hb, this reaction is fastest in the α subunit. PEGylation on the α-subunit in A12 did not prevent access of ascorbate to the ferryl heme, although there was a decrease in the rate of reduction (decreased Vmax, increased Km). However, there were equivocal effects at the β-subunit (increased Vmax, increased Km).
A1 WildType (β-C93) | A12 α-A19C/β-C93A | A12-PEG | |
---|---|---|---|
*p < 0.05 compared to A12 pre-PEGylated value (determined by Student's t-test for autoxidation and heme release, and via the lack of overlap of 95% confidence intervals of means for non linear curve fitting of ferryl reduction rates). | |||
Autoxidation | 0.052 ± 0.020 min−1 | 0.048 ± 0.010 min−1 | 0.029 ± 0.003 min−1 * |
Heme release | 0.046 ± 0.001 min−1 | 0.040 ± 0.010 min−1 | 0.035 ± 0.007 min−1 * |
Ferryl reduction by ascorbate | |||
High affinity rate (α subunit) | V max 0.026 ± 0.003 s−1 | V max 0.024 ± 0.001 s−1 | V max 0.014 ± 0.002 s−1 * |
K M 14.6 ± 5.9 μM | K M 4.32 ± 1.15 μM | K M 18.25 ± 7.87 μM * | |
Autoreduction rate (α subunit) | 0.0066 s−1 | 0.0040 s−1 | 0.0057 s−1 |
Low affinity rate (β subunit) | V max 0.015 ± 0.001 s−1 | V max 0.0074 ± 0.0007 s−1 | V max 0.015 ± 0.001 s−1 * |
K M = 205.7 ± 40.1 μM | K M = 47.1 ± 19.0 μM | K M = 417.2 ± 295.1 μM * | |
Autoreduction rate (β subunit) | 0.0013 s−1 | 0.0008 s−1 | 0.0009 s−1 |
Any introduced site-specific PEG-protein construct must pass three tests: the PEGylation reaction must be efficient; it must not significantly diminish the activity of the protein; and it must not introduce unwanted side reactions. Of the protein we tested A12 (Cys93Ala/αAla19Cys) best fulfills these criteria.
The efficiency of the A12 PEGylation reaction is good (70–80% depending on conditions, Fig. 8). This compares well with the variety of novel surface cysteine reactive residues in granulocyte-macrophage colony-stimulating factor, where efficiencies varies from 45–89%.33 It also compares favorably with the 8–72% yields for the 11 mutations tested in human thyroid stimulating hormone.34 It is also significant that increasing the PEG:Hb ratio above 3:1 and increasing the time of incubation greater than 1 hour had relatively minor effects on this efficiency. This is important, given that grams of PEGylated Hb would be required in any final HBOC product and thus production costs would be a significant fraction of the cost of any commercial product. As a comparison the Hemospan MP4 HBOC was produced with a 20:1 PEG:Hb ratio.21 The natural βCys93 site (A1) is more exposed and reactive in the R state (favored in oxyHb) whereas it is more buried in the T state (favored in deoxyHb).30 However, PEGylation efficiency in A12 is independent of the T or R quaternary state (Fig. 5). This gives additional flexibility in the production of PEGylated A12, which can use either anaerobic or aerobic conditions; in the latter scenario as CO does not hinder PEGylation, it could be added to prevent any oxidation that might occur during processing.
DTT treatment increased the PEG reactivity in some of our preparations, suggesting the presence of disulfide bridges between the introduced cysteine residues. SEC analysis also revealed the presence of a fraction of covalent tetrameric state for both A12 and A13 (Fig. 6). The covalent tetramer could be formed between two dimers of the same Hb molecule, or from two tetramers; these tetramers would then subsequently dissociate into a hybrid tetramer. This has been shown previously with Hb Polytaur (α-Cys104Ser/ α-Ser9Cys human HbA – β-Cys93Ala bovine Hb), although in that case, the formation of a disulfide bridge between two tetramers resulted in the formation of a cyclic trimer of linked tetramers in a time-dependent oxidation process.35 However, disulfide bridges cannot completely explain the small amount of DTT resistant Hb dimer formation that is seen in denaturing SDS-PAGE gels (Fig. 4). It is likely these are due, at least in part, to metal-catalyzed oxidative deamination at the β-subunit36 occurring during the recombinant expression. Consistent with this band excision followed by digestion and peptide mass fingerprinting revealed the presence of predominantly β-subunit. It is clear therefore that, although the A12 mutation appears an ideal candidate for site-specific PEGylation of Hb, more work will be needed to improve the purification process.
Although Hb has a range of activities,37–39 we focused on its primary role in oxygen transport and metabolism. The most exhaustive studies on the effects of site directed mutations on the oxygen affinity of myoglobin (Mb) and Hb have been undertaken by Olson and co-workers.40 They studied over 300 mutants and demonstrated two classes of mutations with significant effects: those that cause local structural changes in the distal heme pocket that change the rate of oxygen entry/exit to/from the heme or alter of the stability of the iron–oxy bond; and those outside the heme pocket near the α1β1 and α2β2 interface that cause global structural perturbations by disrupting the equilibrium between the R (high affinity) and T (low affinity) quaternary states of Hb.41 These active site and allosteric mutations acting independently and in combination have been used to design Hb molecules with P50 values that range from 0.2 to 200 μM, using both allosteric and active site mutations.42 The cysteines we introduced were chosen to be outside the heme pocket and distant from the α1β1 and α2β2 interface, enabling PEGylation that had no effect on both oxygen affinity and cooperative oxygen binding.
To our knowledge the PEGylations on A12 (αAla19Cys) and A13 (βAla13Cys) are the only ones shown to have no significant effect on both oxygen affinity and co-operativity. There is no consensus for the ideal optimum oxygen affinity of an extracellular HBOC. A higher affinity might better deliver oxygen to severely hypoxic tissues, whereas a lower, more physiological, affinity might be better for bulk oxygen transport. These differing strategies are exemplified43 by the higher oxygen affinity (5 Torr) for Sangart's MP4 product, versus values similar to native human Hb for Northfield's Polyheme product (26 Torr) or the lower than native affinities in Biopure's (now HbO2 Therapeutics) Hemopure® product (40 Torr). In this paper we measured oxygen affinities under “pseudophysiological” conditions, similar to those existing outside the red blood cell where high concentrations of physiological effectors such as BPG are not present. So the affinities we measured are lower than those cited above. However, the key fact is that PEGylation of the A12/A13 mutations had no effect on oxygen affinity. These mutations can therefore be used as a template to design other desirable properties without the final PEGylation step modifying oxygen affinity.
As well as mutations specifically designed to modify oxygen affinity,42 other desirable properties that could be added to the A12 template could include adding mutations in the heme pocket to decrease NO scavenging,44 increase ferryl/ferric reduction45,46 or increase nitrite reductase activity.45 Of potential concern is that in order to engineer homogenous PEGylation at a single site we modified the native reactive cysteine residue, β93. The nitrosation of this site has been considered essential for red cell hypoxic signaling,47 though the extent of this effect is controversial.48,49 Our view is that the removal of this reactive residue in extracellular Hb, by replacing it with alanine, is desirable given its tendency to oxidize and cause damage in other HBOCs.50 It is also true that any HBOC infused will still be a minority of the total blood Hb compared to that remaining in the red cell (and which will obviously have intact, reactive βCys93). Therefore, in terms of nitric oxide metabolism we feel the priority in the design of an extracellular HBOC is to decrease the Hb nitric oxide dioxygenase activity and/or increase the nitrite reductase activity. However, we note that, if desired,19 it might be possible to partially protect βCys93 reactivity when PEGylating at α19Cys or β13Cys, depending on the reaction conditions chosen. Clearly this would not be an option if βCys93 itself was chosen as the PEGylation site.
Given its ease of PEGylation and the lack of effect on favorable oxygen binding, we chose to explore further the reactivity of A12 (Cys93Ala/αAla19Cys) in terms of its stability and oxidative reactivity, focusing on heme loss and the autoxidation of ferrous(oxy) Hb to ferric(met)Hb. Heme loss was studied from the ferric Hb as this rate is far greater than the loss from the ferrous redox state51 and is likely to be the physiologically relevant rate for an HBOC in vivo.51,52 The effect of mutations on the rate of globin autoxidation is less well characterized than effects on oxygen affinity.40,42 Although some globins with high oxygen affinity have very low autoxidation rates,53 no Hb mutations that would be able to mirror these effects have been engineered. In general Hb and Mb modifications have no effect or cause an increase in oxidation rate54 and heme loss.42,55 The βCys93Ala/αAla19Cys double mutation itself had no effect on the rate of autoxidation or heme loss, but interestingly, and promisingly, PEGylation at α19Cys decreased both autoxidation and heme loss. This contrasts with PEGylation at βCys93 which elicits the opposite effects.17
It is not easy a priori to predict which surface sites are most efficient for PEGylation in any protein;31 there is always some trial and error involved. However, we conclude that it is indeed possible to engineer an efficient site-specific mutation for PEGylation in human Hb that has no effect on oxygen binding properties and an improvement in the potentially damaging properties of autoxidation and heme loss. PEGylation of Hb at the βCys93Ala/αAla19Cys may therefore prove a useful template as a component of a novel HBOC especially if additional mutations, such as those that decrease nitric oxide scavenging or oxidative stress, are added.
Hb | Hemoglobin |
Mb | Myoglobin |
HBOC | Hemoglobin based oxygen carrier |
A0 | Native human Hb |
A1 | Wild type recombinant human Hb |
A11 | Recombinant human Hb with βCys93Ala mutation |
A12 | Recombinant human Hb with βCys93Ala and αAla19Cys mutations |
A13 | Recombinant human Hb with βCys93Ala and βAla13Cys mutations |
metHb | Met(ferric) hemoglobin |
oxyHb | Oxygenated hemoglobin |
HbCO | Carbon monoxide bound Hb |
SW | Sperm whale |
PEG | Poly(ethylene glycol) |
MAL-PEG | Maleimide-PEG |
PHP | Pyridoxalated hemoglobin polyoxyethylene conjugate |
PMB | Sodium p-hydroxy mercury benzoate |
SEC | Size exclusion chromatography |
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9bm01773a |
This journal is © The Royal Society of Chemistry 2020 |