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Synthesis of a Hemoglobin Polymer ContainingAntioxidant Enzymes Using Complementary

Chemistry of Maleimides and Sulfhydryls

Eugene Tarasov, Melanie M. Blaszak, Jacqueline M. LaMarre, and

Kenneth W. OlsenLoyola University Chicago, Department of Chemistry, Chicago, IL, USA

Abstract: To increase the overall size of hemoglobin (Hb), we developed a novelsystem of polymerization based on the complementary chemistry between sulfhy-dryls and maleimides. The maleimides were introduced onto the protein throughN-(-maleimidobutyryloxy) succinimide, while the sulfhydryls were added using 2-iminothiolane hydrochloride (Trauts reagent). Resulting polymers showedSDS-PAGE bands with molecular weights as high as 96 kDa. Size exclusionchromatography has demonstrated species with molecular weight > 700kDa.The flexibility of the sulfhydryl-maleimide chemistry has also allowed insertionof two antioxidant enzymes, catalase (Cat) and superoxide dismutase (SOD), intothe Hb polymer. Cat was incorporated into the heavier fractions of the polymer,while SOD was found throughout the molecular weight range.

Keywords: 2-iminothiolane; Catalase; Hemoglobin polymer; Maleimide; Super-oxide dismutase

INTRODUCTION

In recent years, evidence has been obtained that suggested that increasedmolecular weight of hemoglobin (Hb)-based blood substitutes mayavoid vasoconstriction [15]. Unmodified Hb and its cross-linked

We would like to thank Kieran P.M. Normoyle and Jennifer E. Dulle for theirhelp on this project. Furthermore, we would like to thank the Hemoglobin Dis-cussion Group at Loyola University Chicago Department of Chemistry for their

Artificial Cells, Blood Substitutes, and Biotechnology , 35: 3143, 2007Copyright Q Informa HealthcareISSN: 1073-1199 print/1532-4184 onlineDOI: 10.1080/10731190600974434

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bis(3,5-dibromosalicyl) fumarate (DBSF)-Hb counterpart, showedincreased vascular resistance through vasoconstriction [6,7]. This effectwas partially attributed to scavenging of the vascular relaxation factornitric oxide (NO) by the Hb. Specifically, it is believed that Hb is ableto come within a close proximity of the interior NO-rich arterial tissueallowing for the binding of the relaxation factor to the protein [8]. Otherstudies suggest that factors such as mechanical forces and S -nitrosylationare also involved in vasoconstriction [9,10].

Glutaraldehyde polymerized-Hb has been used to minimize vasocon-striction in clinical trials [11,12]. The success of these products may occurby physically preventing the migration of the protein species into thearterial wall, thereby preventing diffusion of NO into the Hb. The successof these polymers has allowed for broader clinical trials and licensing insome countries.

Although glutaraldehyde polymerized Hbs are showing promise asblood substitutes, their safety has been questioned [13]. The synthesisof these polymers involves the formation of a Schiff base (imine) betweenthe glutaraldehyde and lysine side chains of the Hb, followed by areduction step using sodium borohydride [14]. Unless it is thoroughlyreduced to an amine, this bond can dissociate, resulting in the breakupof the protein polymer and the release of glutaraldehyde and Hb intothe surrounding environment [13,15].

Another approach that has had significant success is pegylation of Hb. This strategy is based on attachment of large polyethylene glycols(PEG) to the surface of the Hb. While the PEGs do not even doublethe molecular mass (total weight 100 kDa), they do greatly increasethe hydrodynamic volume [16]. It is believed that this modification canminimize vasoconstriction by limiting O 2 delivery to arterial walls, pre-venting O 2 -induced autoregulatory vasoconstriction [17]. Its efficacyhas been demonstrated in clinical trials, but some minor problems havebeen reported [18].

One undesired side effect that is inherent with the use of Hb-basedblood substitutes is iron redox chemistry. Within the red blood cell(RBC), this redox chemistry is controlled by a number of enzymes.Extraction of Hb from the RBC results in oxidation of the iron, whichin some cases is exacerbated by the modification of Hb in the processof making a blood substitute. Previously, it has been shown that theuse of catalase (Cat) and superoxide dismutase (SOD) can decrease theautoxidation rate of Hb [19]. These findings suggest that exogenous anti-oxidant enzymes can be used to restore some of the redox chemistry that

32 E. Tarasov et al.

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chemistry between sulfhydryl and maleimide groups. These groups areintroduced onto the proteins through commercially available com-pounds. The resultant polymer species ranged in molecular weights fromapproximately 100 kDa to > 700 kDa. The flexibility of this system hasalso allowed us to incorporate antioxidant enzymes Cat and SOD intothe polymer, giving the product an ability to combat undesired redoxchemistry.

MATERIALS AND METHODS

Materials

Expired human blood was obtained from LifeSource blood banking ser-vices (Glenview, Illinois). N-(-maleimidobutyryloxy) succinimide (GMBS),2-iminothiolane hydrochloride (Trauts reagent), N-ethylmaleimide (NEM),sodium 2-mercaptoethanesulfonate (MESNA), catalase, superoxide dismu-tase, equine heart cytochrome C, and xanthine were obtained fromSigma-Aldrich (Milwaukee, Wisconsin). Xanthine oxidase was obtainedfrom Calbiochem (La Jolla, California).

Methods

Hemoglobin Purification : The method of Hanash and Shapiro was modi-fied for the purification of Hb [20]. In summary, washed RBCs were lysedusing deionized water, after which Hb was isolated by precipitation of celldebris with ammonium sulfate and centrifugation at 11,000 g. Theisolated Hb was then purified using Pharmacia A KTA Prime equippedwith a Pharmacia Resource 15Q column using a linear sodium chloridegradient.

Maleimide Modification of Proteins : Prior to any modifications, allprotein solutions (i.e., Hb, Cat, and SOD) were extensively dialyzed in50 mM MOPS =10 mM EDTA, pH 7.5. GMBS in dimethyl sulfoxide(DMSO) was then added to the proteins at variable molar ratios. Theprotein solutions, after a 30 min incubation period, were separately dia-lyzed overnight against 50 mM MOPS =10 mM EDTA, pH 7.0. Afterthe buffer exchange, the activated proteins were immediately used in latersteps.

Sulfhydryl Modification of Proteins : Prior to any modifications, allprotein solutions (i.e., Hb, Cat, and SOD) were extensively dialyzed in

Novel Hemoglobin Polymer 33

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dialyzed against 50 mM MOPS =10 mm EDTA pH 7.0. The activated pro-teins were immediately used after buffer exchange.

Polymer formation and capping : Three distinct polymers were made:a) Hb only; b) Hb-Cat; and c) Hb-Cat-SOD. The Hb-polymer wasmade by addition of complementary activated Hb species (i.e., Hb-Mal and Hb-SH), followed by a 30 min incubation period at room tem-perature. After the incubation, non-conjugated free sulfhydryl or malei-mide groups were sequentially capped with NEM and MESNA,respectively, at a 1.1 molar ratio to the initial amount of Trauts reagentand GMBS. For optimal results, NEM in DMSO was added first, fol-lowed by either a dialysis against 50 mM MOPS =10 mM EDTA, pH 7.0for 4 hours or use of a Sephadex G-25 (Pharmacia) desalting columnwith the above mentioned buffer. MESNA, dissolved in a buffersolution, was added to the polymer and after a 30 min incubationperiod the solution was dialyzed (Sephadex G-25 desalting columncan be substituted) against 50 mM MOPS, pH 7.4.

The Hb-Cat polymer was made by first adding complementary acti-vated Hb and Cat, followed by an incubation period. Then Hb activatedwith a complementary group to that of initial Hb was added to the sol-ution. For example, if Hb-SH and Cat-Mal were added first, Hb-Malwas added after the incubation period. On the other hand, if Hb-Maland Cat-SH were added first, Hb-SH would follow the incubation period.Capping was carried out as described for the Hb polymer.

The final polymer made was Hb-Cat-SOD. This product was madeby addition of complementary activated Hb and Cat. After an incubationperiod of 30 min, a solution of similarly activated SOD and Hb wasadded to the Hb-Cat solution. For example, if Hb-SH and Cat-Mal wereadded first, a solution of Hb-Mal and SOD-Mal was then added (Type Ipolymer). Similarly, if Hb-Mal and Cat-SH were reacted first, after anincubation period, a solution Hb-SH and SOD-SH was added (Type IIpolymer). Capping of these types of polymers was accomplished asdescribed for the Hb polymer.

Size Exclusion Chromatography : Size exclusion chromatography(SEC) was conducted using Agilent 1100 modular HPLC utilizing Phe-nomenex BioSep-SEC-S3000 (300 7.80 mm) or Pharmacia C16 =70 col-umn packed with Sephacryl S-300 High Resolution resin. Eluant wasmonitored at a wavelength 280 nm, and the produced chromatogramswere analyzed by ChemOffice V. 8.03 software.

Enzyme Activity Assays : All activity assays were conducted usingAgilent 8453 spectrophotometer utilizing a cell stirrer. Catalase activity


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RESULTS

Hemoglobin Polymer

Modification and capping of the proteins were accomplished using com-mercially available compounds depicted in Fig. 1. Unlike Trauts reagentand MESNA, GMBS and NEM are not soluble in water, therefore theseagents were introduced in DMSO. The volume of DMSO with GMBSor NEM was usually 1.01.5 % of the total protein solutions volume.


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In cases when it was unavoidable to use larger volumes of DMSO, thesolution was added sequentially in small volumes with continuous stir-ring.

The pH of the protein solution was critical in the preparation of thispolymer. The optimal pH for the modification of Lys by Trauts reagent isbetween 79, but at a higher pH undesired reactions with aliphatic andphenolic hydroxyl groups can occur [2326]. To avoid these problems,the reactions were carried out at pH 7.5.

Maleimido modifications of the proteins were done at a slightly basicpH, which ensured that the N -hydroxysuccinimidyl group of GMBS wasmore reactive with the amino side chain of Lys. It is possible to carry outthis type of modification in the pH range of 79, but the hydrolysis of N -hydroxysuccinimidyl increases in basic environments [27]. For optimalresults, all GMBS modifications were carried out at pH 7.5.

Conjugation reactions, on the other hand, were conducted at a neutralpH, which produced larger polymers as monitored by SDS-electro-phoresis. Maleimides have a higher preference for reaction with sulfhy-dryls between pH 6.57.5 [28]. Polymerizations that were carried out atpH 7.5 had lower yields in comparison to the species produced at pH7.0 as monitored by SDS-PAGE (Fig. 2, Lanes 2 and 1, respectively).

Figure 2. SDS-Page of Hb-polymers: Lanes 1 and 2, Hb polymers formed fromequimolar amounts of Hb-Mal and Hb-SH. Lane 1, Hb polymer formed at pH7.0; Lane 2, Hb polymer formed at pH 7.5. Lane 38, species denatured in the

presence of 2-mercaptoethanol; Lane 3, pure Hb; Lane 4, Hb-Mal only; Lane57, Hb polymer species, as lane number increases the amount of Hb-Mal usedfor polymerization decreases; Lane 8 Hb-SH only Lanes 9 14 species denatured


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Polymer yields decreased as the time between activation andconjugation increased. In the case of GMBS modified species, prolongedstorage in an aqueous environment converts the maleimide to itsnon-sulfhydryl reactive acid derivative. Prolonged storage of Trautsmodified proteins increases the possibility of disulfide bridge formation,either intra- or intermolecularly, due to the increased number of sulfhy-dryls on the protein. Disulfide formation decreases the number of freesulfhydryls that can participate in the polymerization. A large molar ratioof Trauts reagent to protein exacerbated this problem.

It is believed that disulfide formation is occurring between Hb-SHs,but these bridges do not significantly add to the formation of the entirepolymer. Gel band separation in the absence of 2-mercaptoethanol (2-ME) produced high molecular weight species (Fig. 2, lane 13), butaddition of 2-ME to the same sample gave only one band at 16 kDa(Fig. 2, lane 8). To rule out the formation of a polymer through disulfideformation, Hb-SH was evaluated by SEC, which showed only one peak( 64 kDa) for Hb-SH, but the peak lacked symmetry, indicating hetero-geneity of the sample.

To determine if polymerization occurred through disulfide cross-linking, polymers were examined by SDS-PAGE in the presence orabsence of 2-ME (Fig. 2, lanes 314). In the presence of 2-ME, the gelprofile produced slightly fewer heavier bands than identical samplesdenatured in its absence (e.g., lanes 57 vs. lanes 1012, Fig. 2).

Verification of polymer formation was conducted by SDS-PAGE,which revealed species with denatured molecular weights from 16to 100 kDa (Fig. 2). The yields of higher molecular weight speciesdecreased as the amount of maleimides decreased, as quantified by thelower intensity of gel bands (data not shown). Under denaturing con-ditions the band distribution of Hb-Mal (lanes 4 and 9) and Hb-SH (lane13) separately gave species with molecular weights as high as 70 kDa and32 kDa, respectively. In the case of Hb-Mal, polymer formation was ruledout by SEC, which showed that Hb-Mal eluted at the same time as non-modified Hb. This finding indicates that any cross-linking formed in Hb-Mal had to be intramolecular.

To explore the molecular weight distribution of the polymers, SECwas utilized under native conditions. Highly modified Hbs when mixedat equimolar amounts produced species that favored higher molecularweight species (> 700 kDa) (Fig. 3). When the activated Hbs were mixedat non-equimolar amounts, there was preference for formation of mid-weight species ( 300 kDa) unless the Hbs were highly modified, which


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were modified at molar equivalent of greater than 30 to 1 (modifying spe-cies to Hb tetramer). Optimization of the polymerization conditionshowed that Hb modified at a molar ratio of 1525 (modifying agentto the Hb) and mixed at equimolar amounts produced heavy polymerswithout any noticeable precipitation.

HEMOGLOBIN-ENZYME POLYMERS

We also used the sulfhydryl-maleimide system to incorporate the antiox-

Figure 3. Size exclusion chromatography of polymerized human Hbs: a) Purenon-modified Hb; b) Hb complex resulting from addition of Hb-(10)-SH andHb-(10)-Mal added at equal molar ratios; c) complex resulting from Hb-(15)-SH and Hb-(15)-Mal; d) complex resulting from Hb-(25)-SH and Hb-(25)-Mal.The number before the -SH refers to the mole excess of Trauts reagent used tomodify the Hb. The number before the -Mal refers to the mole excess of GMBS

used to modify the Hb.


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volume. In addition, SDS-PAGE gel distribution of the heterogeneouspolymers did not differ significantly from that of the Hb-only polymer(unpublished results). Since the SDS-PAGE gel band distributions of Cat and SOD are very similar to that of polymerized Hb, it is difficultto evaluate if the cross-linking is occurring between the enzymes or Hb.

The incorporation of either Cat or SOD into the Hb polymer wasevaluated using enzyme specific activity assays. The polymers were frac-tionated using SEC, and each fraction was assayed for the presence of both Cat and SOD (Fig. 4). In all cases, Cat activity was found in the

Figure 4. SEC of heterogeneous Hb-Cat-SOD polymers obtained using Pharma-cia Sephacryl S-300: a) Type I polymer Polymer made by first conjugating Cat-


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heavier end in both Hb-Cat (data not shown) and Hb-Cat-SOD polymer.On the other hand, SOD activity was found throughout the Hb-Cat-SODpolymer. To rule out the possibility of Cat or SOD individually formingpolymers, SEC of activated species was conducted, which showedretention times similar to that of non-modified species but with non-symmetrical peaks, possibly indicating a small amount of intermolecularcross-linking (data not shown).

Two distinct Hb-Cat-SOD polymers were made which differed in theorder of polymerization. The first type of polymer was made by combin-ing Cat-Mal and Hb-SH followed by an addition of Hb-Mal and SOD-Mal (Type I). The second polymer was made by combining Cat-SHand Hb-Mal, followed by addition of Hb-SH and SOD-SH (Type II).Cat activity within both types of polymers was found within the highmolecular weight polymers (Fig. 4). The middle and smaller weight poly-mers also displayed Cat activity but the magnitude was significantlysmaller. Although SOD activity was found throughout the polymers,there were differences between Type I and Type II polymers. In the TypeI polymer (Fig. 4a), the highest activity was found at the heavier- to mid-dle- weight polymers. Type II polymers (Fig. 4b) did not have any specificpreference, and the SOD activity was found throughout the polymer atsimilar magnitudes.

DISCUSSION

One of the concerns of these syntheses is possible polymer formationthrough disulfide bonds, but this was not a significant problem becausethey do not dominate the reaction. Comparison of SDS-PAGE bandsof polymers denatured in the presence of a disulfide reducing agent doesnot differ significantly from the separation achieved with a denaturingsolution that lacked 2-ME (Fig. 2, lanes 314). This indicates that thepolymer is formed primarily by sulfo-maleimido coupling. Treatmentof the retained polymer by a 100 kDa diafiltration membrane with 2-ME did not release Hb from the retantate. Therefore, formation of dis-ulfide bonds should not pose a risk of polymer degradation.

The distribution of these polymers on SEC appears to be very similarto that of glutaraldehyde-polymerized Hb [29]. As in the case of glutar-aldehyde-polymerized Hb, there is no single species that can be identified.This finding, along with the data from SDS-PAGE, suggests that the


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There is a clear preference for higher molecular weight species whenhighly modified Hbs are reacted. Furthermore, use of slightly modifiedHbs produced the opposite effect, creating smaller weight species. Properselection of reaction conditions is very important, since use of highlymodified species (i.e., > 30 to 1 modifying species to Hb) producedprecipitate, while use of lightly modified species gave poor yields. Foroptimal results, the data suggest use of moderately modified Hbs. Theseare the Hbs that have been modified at a molar ratio between 1525 witheither GMBS or Trauts to Hb. Furthermore, the data also suggest thatmixing of equimolar amounts of activated Hbs produced good yields,while having excess of one species over another did not prove to beadvantageous in the formation of heavier species unless they were heavilymodified.

Complementary chemistry can be applied to introduce other proteinsinto the Hb polymers with some control over the formation of the species.In the Hb-Cat-SOD species, the majority of the Cat activity was found atthe heavier end of the eluted material (Fig. 4). SOD activity, in comparisonto Cat activity, had a significantly larger spread. Cat is a large globular pro-tein, with a large surface area. Reacting complementary modified Cat andHb allows Cat to function as a scaffold to which Hb attaches. Since not allactive groups on the Hb, either maleimides or sulfhydryls, are participatingin coupling between Cat and Hb, additional Hb and =or SOD can be added.The only limitation to the last step in this synthesis is that the protein mustbe activated with a group that is complementary to the first Hb. The result-ing species presumably resembles a dendrite with Cat in the center and Hbpolymers as dendritic arms (Fig. 5).

Figure 5 Possible structure of a heterogeneous polymer =dendrite According to


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CONCLUSION

The goal of this study was to develop a new system for formation of a Hbpolymer that does not use glutaraldehyde. The results show that a Hbpolymer can be formed based on the complementary chemistry betweensulfhydryls and maleimides. In addition, this system is very flexible andis capable of accepting other proteins without loss of polymerization.

The flexibility and the framework of this type of system is beinginvestigated in other areas. For instance, efforts are being applied toinvestigate the feasibility of using other types of Hb sources for polymer-ization. Early results indicate that a bovine Hb polymer can be made toproduce similar results to that of a human Hb polymer analogue. Inaddition, other capping reagents such as maleimido-polyethyleneglycolare being investigated as an alternative to the currently used species.

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