Artificial Cells, Blood Substitutes, and Biotechnology, 35: 3143, 2007 Copyright Q Informa Healthcare ISSN: 1073-1199 print/1532-4184

online DOI: 10.1080/10731190600974434

Synthesis of a Hemoglobin Polymer Containing Antioxidant Enzymes Using Complementary Chemistry of Maleimides and Sulfhydryls
Eugene Tarasov, Melanie M. Blaszak, Jacqueline M. LaMarre, and Kenneth W. Olsen
Loyola University Chicago, Department of Chemistry, Chicago, IL, USA

Abstract: To increase the overall size of hemoglobin (Hb), we developed a novel system of polymerization based on the complementary chemistry between sulfhydryls and maleimides. The maleimides were introduced onto the protein through N-(-maleimidobutyryloxy) succinimide, while the sulfhydryls were added using 2iminothiolane hydrochloride (Trauts reagent). Resulting polymers showed SDS-PAGE bands with molecular weights as high as 96 kDa. Size exclusion chromatography has demonstrated species with molecular weight > 700 kDa. The flexibility of the sulfhydryl-maleimide chemistry has also allowed insertion of two antioxidant enzymes, catalase (Cat) and superoxide dismutase (SOD), into the 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; Superoxide dismutase

INTRODUCTION In recent years, evidence has been obtained that suggested that increased molecular weight of hemoglobin (Hb)-based blood substitutes may avoid vasoconstriction [15]. Unmodified Hb and its cross-linked
We would like to thank Kieran P.M. Normoyle and Jennifer E. Dulle for their help on this project. Furthermore, we would like to thank the Hemoglobin Discussion Group at Loyola University Chicago, Department of Chemistry, for their valuable comments. This work was partially supported by U.S. Department of Education GAANN fellowship (P200A030223) and NSF REU Grant (0243825). Address correspondence to Kenneth W. Olsen. E-mail: kolsen@iac.edu 31

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bis(3,5-dibromosalicyl) fumarate (DBSF)-Hb counterpart, showed increased vascular resistance through vasoconstriction [6,7]. This effect was partially attributed to scavenging of the vascular relaxation factor nitric oxide (NO) by the Hb. Specifically, it is believed that Hb is able to come within a close proximity of the interior NO-rich arterial tissue allowing for the binding of the relaxation factor to the protein [8]. Other studies suggest that factors such as mechanical forces and S-nitrosylation are also involved in vasoconstriction [9,10]. Glutaraldehyde polymerized-Hb has been used to minimize vasoconstriction in clinical trials [11,12]. The success of these products may occur by physically preventing the migration of the protein species into the arterial wall, thereby preventing diffusion of NO into the Hb. The success of these polymers has allowed for broader clinical trials and licensing in some countries. Although glutaraldehyde polymerized Hbs are showing promise as blood substitutes, their safety has been questioned [13]. The synthesis of these polymers involves the formation of a Schiff base (imine) between the glutaraldehyde and lysine side chains of the Hb, followed by a reduction step using sodium borohydride [14]. Unless it is thoroughly reduced to an amine, this bond can dissociate, resulting in the breakup of the protein polymer and the release of glutaraldehyde and Hb into the 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 double the molecular mass (total weight  100 kDa), they do greatly increase the hydrodynamic volume [16]. It is believed that this modification can minimize vasoconstriction by limiting O2 delivery to arterial walls, preventing O2-induced autoregulatory vasoconstriction [17]. Its efficacy has been demonstrated in clinical trials, but some minor problems have been reported [18]. One undesired side effect that is inherent with the use of Hb-based blood 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, which in some cases is exacerbated by the modification of Hb in the process of making a blood substitute. Previously, it has been shown that the use of catalase (Cat) and superoxide dismutase (SOD) can decrease the autoxidation rate of Hb [19]. These findings suggest that exogenous antioxidant enzymes can be used to restore some of the redox chemistry that is afforded by the RBC. As an alternative to glutaraldehyde polymerized and pegylated Hbs, we present a system for Hb polymerization based on the complementary

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chemistry between sulfhydryl and maleimide groups. These groups are introduced onto the proteins through commercially available compounds. The resultant polymer species ranged in molecular weights from approximately 100 kDa to > 700 kDa. The flexibility of this system has also allowed us to incorporate antioxidant enzymes Cat and SOD into the polymer, giving the product an ability to combat undesired redox chemistry. MATERIALS AND METHODS Materials Expired human blood was obtained from LifeSource blood banking services (Glenview, Illinois). N-(-maleimidobutyryloxy) succinimide (GMBS), 2-iminothiolane hydrochloride (Trauts reagent), N-ethylmaleimide (NEM), sodium 2-mercaptoethanesulfonate (MESNA), catalase, superoxide dismutase, equine heart cytochrome C, and xanthine were obtained from Sigma-Aldrich (Milwaukee, Wisconsin). Xanthine oxidase was obtained from Calbiochem (La Jolla, California). Methods Hemoglobin Purification: The method of Hanash and Shapiro was modified for the purification of Hb [20]. In summary, washed RBCs were lysed using deionized water, after which Hb was isolated by precipitation of cell debris with ammonium sulfate and centrifugation at 11,000 g. The KTA Prime equipped isolated Hb was then purified using Pharmacia A with a Pharmacia Resource 15Q column using a linear sodium chloride gradient. Maleimide Modification of Proteins: Prior to any modifications, all protein solutions (i.e., Hb, Cat, and SOD) were extensively dialyzed in 50 mM MOPS=10 mM EDTA, pH 7.5. GMBS in dimethyl sulfoxide (DMSO) was then added to the proteins at variable molar ratios. The protein solutions, after a 30 min incubation period, were separately dialyzed overnight against 50 mM MOPS=10 mM EDTA, pH 7.0. After the buffer exchange, the activated proteins were immediately used in later steps. Sulfhydryl Modification of Proteins: Prior to any modifications, all protein solutions (i.e., Hb, Cat, and SOD) were extensively dialyzed in 50 mM MOPS=10 mm EDTA, pH 7.5. Trauts reagent was then added to the proteins at variable molar ratios in 50 mM MOPS=10 mM EDTA, pH 7.5. After a 30 min incubation period, all solutions were separately

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dialyzed against 50 mM MOPS=10 mm EDTA pH 7.0. The activated proteins 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 was made by addition of complementary activated Hb species (i.e., HbMal and Hb-SH), followed by a 30 min incubation period at room temperature. After the incubation, non-conjugated free sulfhydryl or maleimide groups were sequentially capped with NEM and MESNA, respectively, at a 1.1 molar ratio to the initial amount of Trauts reagent and GMBS. For optimal results, NEM in DMSO was added first, followed by either a dialysis against 50 mM MOPS=10 mM EDTA, pH 7.0 for 4 hours or use of a Sephadex G-25 (Pharmacia) desalting column with the above mentioned buffer. MESNA, dissolved in a buffer solution, was added to the polymer and after a 30 min incubation period the solution was dialyzed (Sephadex G-25 desalting column can be substituted) against 50 mM MOPS, pH 7.4. The Hb-Cat polymer was made by first adding complementary activated Hb and Cat, followed by an incubation period. Then Hb activated with a complementary group to that of initial Hb was added to the solution. For example, if Hb-SH and Cat-Mal were added first, Hb-Mal was added after the incubation period. On the other hand, if Hb-Mal and 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 made by addition of complementary activated Hb and Cat. After an incubation period of 30 min, a solution of similarly activated SOD and Hb was added to the Hb-Cat solution. For example, if Hb-SH and Cat-Mal were added first, a solution of Hb-Mal and SOD-Mal was then added (Type I polymer). Similarly, if Hb-Mal and Cat-SH were reacted first, after an incubation period, a solution Hb-SH and SOD-SH was added (Type II polymer). Capping of these types of polymers was accomplished as described for the Hb polymer. Size Exclusion Chromatography: Size exclusion chromatography (SEC) was conducted using Agilent 1100 modular HPLC utilizing Phenomenex BioSep-SEC-S3000 (300 7.80 mm) or Pharmacia C16=70 column packed with Sephacryl S-300 High Resolution resin. Eluant was monitored at a wavelength 280 nm, and the produced chromatograms were analyzed by ChemOffice V. 8.03 software. Enzyme Activity Assays: All activity assays were conducted using Agilent 8453 spectrophotometer utilizing a cell stirrer. Catalase activity was determined by the method described by Beer and Sizer [21]. SOD activity assay was conducted by the method of McCord and Fredovich [22].

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RESULTS Hemoglobin Polymer Modification and capping of the proteins were accomplished using commercially available compounds depicted in Fig. 1. Unlike Trauts reagent and MESNA, GMBS and NEM are not soluble in water, therefore these agents were introduced in DMSO. The volume of DMSO with GMBS or NEM was usually 1.01.5% of the total protein solutions volume.

Figure 1. Modification of proteins and capping of activated groups. Initially, proteins were activated using schemes a and b. After activation and polymerization, non-conjugated active groups were capped using the schemes depicted c and d.

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In cases when it was unavoidable to use larger volumes of DMSO, the solution was added sequentially in small volumes with continuous stirring. The pH of the protein solution was critical in the preparation of this polymer. The optimal pH for the modification of Lys by Trauts reagent is between 79, but at a higher pH undesired reactions with aliphatic and phenolic 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 basic pH, which ensured that the N-hydroxysuccinimidyl group of GMBS was more reactive with the amino side chain of Lys. It is possible to carry out this type of modification in the pH range of 79, but the hydrolysis of N-hydroxysuccinimidyl increases in basic environments [27]. For optimal results, all GMBS modifications were carried out at pH 7.5. Conjugation reactions, on the other hand, were conducted at a neutral pH, which produced larger polymers as monitored by SDS-electrophoresis. Maleimides have a higher preference for reaction with sulfhydryls between pH 6.57.5 [28]. Polymerizations that were carried out at pH 7.5 had lower yields in comparison to the species produced at pH 7.0 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 from equimolar amounts of Hb-Mal and Hb-SH. Lane 1, Hb polymer formed at pH 7.0; 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; Lane 57, Hb polymer species, as lane number increases the amount of Hb-Mal used for polymerization decreases; Lane 8, Hb-SH only. Lanes 914, species denatured in the absence of 2-mercaptoethanol; Lane 9, Hb-Mal only; Lane 1012, Hb polymer species, as lane number increases the amount of Hb-Mal used for polymerization decreases; Lane 13, Hb-SH only; and Lane 14, pure Hb.

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Polymer yields decreased as the time between activation and conjugation increased. In the case of GMBS modified species, prolonged storage in an aqueous environment converts the maleimide to its non-sulfhydryl reactive acid derivative. Prolonged storage of Trauts modified proteins increases the possibility of disulfide bridge formation, either intra- or intermolecularly, due to the increased number of sulfhydryls on the protein. Disulfide formation decreases the number of free sulfhydryls that can participate in the polymerization. A large molar ratio of 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 entire polymer. Gel band separation in the absence of 2-mercaptoethanol (2ME) produced high molecular weight species (Fig. 2, lane 13), but addition 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 disulfide formation, Hb-SH was evaluated by SEC, which showed only one peak ( 64 kDa) for Hb-SH, but the peak lacked symmetry, indicating heterogeneity of the sample. To determine if polymerization occurred through disulfide crosslinking, polymers were examined by SDS-PAGE in the presence or absence of 2-ME (Fig. 2, lanes 314). In the presence of 2-ME, the gel profile produced slightly fewer heavier bands than identical samples denatured 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  16 to  100 kDa (Fig. 2). The yields of higher molecular weight species decreased as the amount of maleimides decreased, as quantified by the lower intensity of gel bands (data not shown). Under denaturing conditions the band distribution of Hb-Mal (lanes 4 and 9) and Hb-SH (lane 13) separately gave species with molecular weights as high as 70 kDa and 32 kDa, respectively. In the case of Hb-Mal, polymer formation was ruled out by SEC, which showed that Hb-Mal eluted at the same time as nonmodified Hb. This finding indicates that any cross-linking formed in HbMal had to be intramolecular. To explore the molecular weight distribution of the polymers, SEC was utilized under native conditions. Highly modified Hbs when mixed at equimolar amounts produced species that favored higher molecular weight species (>700 kDa) (Fig. 3). When the activated Hbs were mixed at non-equimolar amounts, there was preference for formation of midweight species (300 kDa) unless the Hbs were highly modified, which restored the preference for higher weight species. Thus, highly modified species were better choices for greater yields of high molecular weight Hbs, but precipitation was observed for polymers formed from Hbs that

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Figure 3. Size exclusion chromatography of polymerized human Hbs: a) Pure non-modified Hb; b) Hb complex resulting from addition of Hb-(10)-SH and Hb-(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 to modify the Hb. The number before the -Mal refers to the mole excess of GMBS used to modify the Hb.

were modified at molar equivalent of greater than 30 to 1 (modifying species to Hb tetramer). Optimization of the polymerization condition showed that Hb modified at a molar ratio of 1525 (modifying agent to the Hb) and mixed at equimolar amounts produced heavy polymers without any noticeable precipitation. HEMOGLOBIN-ENZYME POLYMERS We also used the sulfhydryl-maleimide system to incorporate the antioxidant enzymes, Cat and SOD, into the Hb-polymer. The SEC chromatograms did not show significant differences between Hb-Cat and Hb-CatSOD to that of Hb-only polymer, since all polymers eluted at the void

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volume. In addition, SDS-PAGE gel distribution of the heterogeneous polymers 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 difficult to evaluate if the cross-linking is occurring between the enzymes or Hb. The incorporation of either Cat or SOD into the Hb polymer was evaluated using enzyme specific activity assays. The polymers were fractionated 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 Pharmacia Sephacryl S-300: a) Type I polymer. Polymer made by first conjugating CatMal and Hb-SH, followed by addition of Hb-Mal and SOD-Mal; b) Type II polymer. Polymer made by first conjugating Cat-SH and Hb-Mal, followed by addition of Hb-SH and SOD-SH.

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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-SOD polymer. To rule out the possibility of Cat or SOD individually forming polymers, SEC of activated species was conducted, which showed retention times similar to that of non-modified species but with nonsymmetrical peaks, possibly indicating a small amount of intermolecular cross-linking (data not shown). Two distinct Hb-Cat-SOD polymers were made which differed in the order of polymerization. The first type of polymer was made by combining Cat-Mal and Hb-SH followed by an addition of Hb-Mal and SODMal (Type I). The second polymer was made by combining Cat-SH and Hb-Mal, followed by addition of Hb-SH and SOD-SH (Type II). Cat activity within both types of polymers was found within the high molecular weight polymers (Fig. 4). The middle and smaller weight polymers also displayed Cat activity but the magnitude was significantly smaller. Although SOD activity was found throughout the polymers, there were differences between Type I and Type II polymers. In the Type I polymer (Fig. 4a), the highest activity was found at the heavier- to middle- weight polymers. Type II polymers (Fig. 4b) did not have any specific preference, and the SOD activity was found throughout the polymer at similar magnitudes.

DISCUSSION One of the concerns of these syntheses is possible polymer formation through disulfide bonds, but this was not a significant problem because they do not dominate the reaction. Comparison of SDS-PAGE bands of polymers denatured in the presence of a disulfide reducing agent does not differ significantly from the separation achieved with a denaturing solution that lacked 2-ME (Fig. 2, lanes 314). This indicates that the polymer is formed primarily by sulfo-maleimido coupling. Treatment of the retained polymer by a 100 kDa diafiltration membrane with 2ME did not release Hb from the retantate. Therefore, formation of disulfide bonds should not pose a risk of polymer degradation. The distribution of these polymers on SEC appears to be very similar to that of glutaraldehyde-polymerized Hb [29]. As in the case of glutaraldehyde-polymerized Hb, there is no single species that can be identified. This finding, along with the data from SDS-PAGE, suggests that the polymer solution is not homogeneous. Instead, it is a heterogeneous solution made up of variably sized oligomers, but the distribution of these oligomers can be controlled through reaction conditions.

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There is a clear preference for higher molecular weight species when highly modified Hbs are reacted. Furthermore, use of slightly modified Hbs produced the opposite effect, creating smaller weight species. Proper selection of reaction conditions is very important, since use of highly modified species (i.e., > 30 to 1 modifying species to Hb) produced precipitate, while use of lightly modified species gave poor yields. For optimal results, the data suggest use of moderately modified Hbs. These are the Hbs that have been modified at a molar ratio between 1525 with either GMBS or Trauts to Hb. Furthermore, the data also suggest that mixing of equimolar amounts of activated Hbs produced good yields, while having excess of one species over another did not prove to be advantageous in the formation of heavier species unless they were heavily modified. Complementary chemistry can be applied to introduce other proteins into 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 at the heavier end of the eluted material (Fig. 4). SOD activity, in comparison to Cat activity, had a significantly larger spread. Cat is a large globular protein, with a large surface area. Reacting complementary modified Cat and Hb allows Cat to function as a scaffold to which Hb attaches. Since not all active groups on the Hb, either maleimides or sulfhydryls, are participating in 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 must be activated with a group that is complementary to the first Hb. The resulting species presumably resembles a dendrite with Cat in the center and Hb polymers as dendritic arms (Fig. 5).

Figure 5. Possible structure of a heterogeneous polymer=dendrite. According to experimental design, activated Cat was first reacted with a complementary activated Hb. After an incubation period, an additional round of Hb and SOD was added.

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CONCLUSION The goal of this study was to develop a new system for formation of a Hb polymer that does not use glutaraldehyde. The results show that a Hb polymer can be formed based on the complementary chemistry between sulfhydryls and maleimides. In addition, this system is very flexible and is capable of accepting other proteins without loss of polymerization. The flexibility and the framework of this type of system is being investigated in other areas. For instance, efforts are being applied to investigate the feasibility of using other types of Hb sources for polymerization. Early results indicate that a bovine Hb polymer can be made to produce similar results to that of a human Hb polymer analogue. In addition, other capping reagents such as maleimido-polyethyleneglycol are being investigated as an alternative to the currently used species.

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