Polymerization of Diaspirin Cross-Linked Hemoglobin (DCLHb) with

Bioconjugate Chem. 1999, 10, 1013−1020

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Polymerization of Diaspirin Cross-Linked Hemoglobin (DCLHb) with Water-Soluble, Nonimmunogenic Polyamide Cross-Linking Agents Ton T. Hai,* David E. Pereira, Deanna J. Nelson,† Ana Srnak, and James Catarello Corporate Research and Technical Services, Baxter Healthcare Corporation, 25212 West State Route 120, Round Lake, Illinois 60073-9799 . Received April 28, 1999; Revised Manuscript Received August 17, 1999

Diaspirin cross-linked hemoglobin (DCLHb), a human hemoglobin that is intramolecularly cross-linked between the R chains (lysine 99R1-lysine 99R2), was polymerized with a number of water-soluble, nonimmunogenic polyamide cross-linking agents. The degree of polymerization and the oxygen-carrying capacity depended upon the polyamide reagent, the starting concentration of DCLHb, the molar ratio of the polyamide reagent to DCLHb used, the reaction pH, and whether oxy- or deoxy-DCLHb was used in the polymerization reaction.

INTRODUCTION

It is generally accepted in the blood substitutes community that increasing the size of hemoglobin by polymerization or surface modification offers the benefits of reduced renal clearance, reduced rate of metabolism, and increased duration in the vascular circulation (1-3). Historically, this was accomplished by using reagents such as glutaraldehyde (4), poly(ethylene glycol) (5), or dextran (6, 7). The polymerized products were mixtures of hemoglobin, oligomers of hemoglobin, and polymers of hemoglobin having a spectrum of sizes and molecular weights. If a product in a specific size range was desired, it had to be separated from the bulk product mixture by chromatographic or ultrafiltration methods. Because the separation of such product mixtures into the various size ranges on a commercial scale is technically difficult, time consuming, and costly, our goal was to control the product composition of hemoglobin polymerization using water-soluble, nonimmunogenic polyamide cross-linking agents (8, 9). These novel cross-linking agents were used to effect the polymerization of diaspirin cross-linked hemoglobin (DCLHb), a human hemoglobin that is intramolecularly cross-linked between the R chains (lysine 99R1-lysine 99R2) (10, 11). The goal of the research was to obtain a polymerized DCLHb (poly-DCLHb) product that would meet the following requirements (a) The polyDCLHb would be an oxygendelivering solution suitable for large-volume infusions. (b) It would have a longer half-life in the systemic circulation as oxyhemoglobin than DCLHb. (c) It would have an enhanced safety profile relative to DCLHb. (d) It would have a stability profile at least equivalent to that of DCLHb. Objectives a-c appeared to be related to the size of the polyDCLHb. We recognized that, in general, larger hemoglobins are retained longer in the vascular system. Moreover, we also recognized those polyhemoglobin molecules that are too large are prone to aggregation and/ or precipitation. With this in mind, polyDCLHb synthesis was directed to obtain a polymerized DCLHb product * To whom correspondence should be addressed. Phone: (847) 270-5837. Fax: (847) 270-5897. E-mail: [email protected]. † Current address: Magellan Laboratories Inc., P.O. Box 13341, Research Triangle Park, NC 27709.

having hemoglobin molecules with molecular mass in the range 130000-260000 Da (equivalent to dimers to tetramers of DCLHb). To this end, we have tried various synthetic approaches in an attempt to produce a polyDCLHb solution that contained neither residual DCLHb nor high molecular mass polymer (defined as any product having a molecular mass exceeding the exclusion limit of 1 × 106 Da of a TSK G4000SW column). These experiments included the assessment of a spectrum of polyamide reagents with varying molecular masses and terminal reactive functional groups, using various concentrations of DCLHb, varying the number of molar equivalents of the polyamide reagent, and altering the reaction pH. EXPERIMENTAL PROCEDURES

General. Diaspirin cross-linked hemoglobin (DCLHb), Plasma Lyte A, and Lactated Ringer’s irrigation solution were obtained from Baxter Healthcare Corporation, Deerfield, IL. The polyamide cross-linking agents were prepared as previously described (8). All other commercial reagents were of the highest purity available. All solvents used were of HPLC grade. Analytical Methods. Analytical RP-HPLC was carried out on a Vydac Protein C4 column (5 µm, 4.6 × 250 mm). The mobile phase was delivered at 1 mL/min as a linear gradient of B (CH3CN:H2O:TFA, 60:40:0.1, by volume) in A (CH3CN:H2O:TFA, 20:80:0.1, by volume) as follows: (1) 50% B to 55% B over 20 min, (2) 55% B to 75% B over 10 min, and (3) 75% B to 85% B over 10 min. Analytes were detected by measuring the absorbance at 220 nm. Size-exclusion chromatography (SEC) was performed using TSK G4000SW and TSK G3000SW columns (7.5 × 300 mm, TosoHaas) connected in series. Analytes were eluted with a binary mobile phase (50 mM phosphate, pH 6.5/2-propanol, 9:1 by volume) delivered at a flow rate of 1 mL/min. Analytes were detected by measuring the absorbance at 280 nm. Oxygen equilibrium curves were recorded by reoxygenation of nitrogen equilibrated deoxyhemoglobin in 0.1 M Bis-Tris buffer (pH 7.4 at 37 °C) in the spectral cuvette of a Hemox Analyzer (TCS Medical Products Co., Huntington Valley, PA). The oxygen pressure at which

10.1021/bc9900486 CCC: $18.00 © 1999 American Chemical Society Published on Web 10/15/1999

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hemoglobin is half saturated (P50) and the subunit cooperativity (n40-60) were calculated from the oxygen equilibrium curves. The concentrations of oxy-, deoxy-, and methemoglobin were calculated from their absorbances at three wavelengths (630, 576, and 560 nm) and the reported molar extinction coefficients (12). Total hemoglobin concentration was calculated by summation of the concentrations of these components, and percent methemoglobin (MetHb) was calculated as its portion of the total hemoglobin concentration. The number of reactive thiols on the polyDCLHb molecules was determined by the method of Neis et al. (13). Polymerization of DCLHb with Activated Esters (1a, 1b, and 2a Reagents). Method 1. DCLHb polymerizations with activated esters of the polyamides were completed as follows. A solution of DCLHb in 0.1 M Hepes, pH 7, or DCLHb in Lactated Ringer’s solution was deoxygenated by successive vacuum/nitrogen cycles at room temperature for 1.5 h. In some experiments, 15 molar equivalents of sodium tripolyphosphate (STP) were added to facilitate hemoglobin deoxygenation. A solution of the activated ester in deoxygenated water was added quickly to the deoxyDCLHb solution, and the resulting reaction solution was stirred at room temperature under nitrogen. The course of the reaction was monitored by SEC. When a suitable SEC profile was observed or the reaction was complete (the point at which the SEC profile no longer changed), the reaction mixture was cooled to 5 °C, and a solution of 1 M N-acetyl-L-cysteine (NAC), pH 9, was added (i.e., NAC/1a, 1b, or 2a molar ratio of 4:1). The solution was stirred at 5 °C under nitrogen overnight, and then the solution was dialyzed against Lactated Ringer’s solution to give the final product. Method 2. A solution of the 1a, 1b, or 2a in cold, sterile water was added through a 0.2 µm pore-size filter to a deoxygenated DCLHb solution in 0.1 M Hepes, pH 8 at 5 °C. In some experiments, 15 molar equivalents of sodium tripolyphosphate were added to facilitate the hemoglobin deoxygenation. After the addition of the polyamide solution, the reaction mixture was stirred at 5 °C under nitrogen, and the course of the polymerization was monitored by SEC. When the reaction was complete, the polyDCLHb solution was reoxygenated, concentrated to 7-7.5 g/dL, and diafiltered into PlasmaLyte A solution. Effects of DCLHb Concentration. Polymerizations of DCLHb with 1a (MW 4200) were completed under the conditions described in method 1. In these reactions, the DCLHb concentration was varied from 3 to 15 g/dL, and the effects of DCLHb concentration on the degree of polymerization were monitored by SEC. The experimental data are summarized in Table 1 and Figure 2. Effects of Increased Polyamide Molecular Mass. Polymerizations of DCLHb (15 g/dL) with 1a (MW 4200) and 1a (MW 7600) were completed as described in method 1. The polymerization was complete in about 1 h for 1a (MW 7600) and in about 4 h for 1a (MW 4200). The experimental data are summarized in Supporting Information, Table S-1, and Figure 3. Effects of Changes in Reaction pH. The polymerization of DCLHb in 0.1 M Hepes, pH 8, or in 0.1 M borate buffer, pH 9, with 1a (MW 5600) or 1b (MW 5200) was performed as follows. A solution of DCLHb (3 g/dL) in 0.1 M borate buffer, pH 9, containing 15 equiv of STP was deoxygenated by vacuum/nitrogen cycles at room temperature for 1.5 h. The deoxygenated hemoglobin solution was cooled to 5 °C, and a deoxygenated solution of 1a (MW 5600) or 1b (MW 5200) in deoxygenated water was added. The solution was stirred at 5 °C under

Hai et al.

Figure 1. Polyamide reagent structures.

nitrogen, and the reaction was monitored by SEC. The polymerization was complete in about 3.0 h. The reaction mixture was dialyzed against PlasmaLyte A solution to give the final product. The experimental results are summarized in Supporting Information, Table S-2. Polymerization of DCLHb with Maleimides [3a (MW 4080)]. A solution of DCLHb (20 g/dL) was deoxygenated by successive vacuum/nitrogen cycles for 1.5 h at room temperature. A solution of the maleimideterminated polyamide reagent 3a (MW 4080) in deoxygenated water was added immediately to the deoxy-DCLHb solution, and the reaction mixture was stirred at room temperature under nitrogen. The course of the reaction was followed by SEC. After 15 h, the reaction was complete. The reaction mixture was cooled to 5 °C and a solution of 1 M N-acetyl-L-cysteine, pH 9 [molar ratio NAC to 3a (MW 4080) of 5] was added. The solution was stirred at 5 °C under nitrogen overnight and then dialyzed against Lactated Ringer’s solution to give the final product. The experimental data are presented in Supporting Information, Table S-3. Polymerization of DCLHb with Aldehydes (4a). A solution of DCLHb (15 g/dL) in 0.1 M Hepes, pH 7,

Polymerization of Diaspirin Cross-Linked Hemoglobin

Figure 2. SEC profiles of 1a (MW 4200) polymerized DCLHb.

was deoxygenated by successive vacuum/nitrogen cycles for 1.5 h at room temperature. A solution of 4a (MW 4000) and a reducing agent (in a molar ratio of reducing agent to 4a of 10) was added. When sodium borohydride was used as the reducing agent, it was added after 20 h to allow time for imine formation and to avoid reducing the aldehyde groups of 4a. After 24 h, the reaction mixture was cooled to 5 °C, and 1.0 M glycine, pH 9 (molar ratio of glycine to 4a of 4), and 1.0 M NAC, pH 9 (molar ratio NAC to DCLHb of 8), were added to quench the polymerization and to reduce methemoglobin, respectively. The solution was stirred under nitrogen at 5 °C overnight and then dialyzed against Lactated Ringer’s solution to give the final products. The experimental results are summarized in Supporting Information, Table S-3. Effects of Polymerization with OxyDCLHb. A solution of oxy- or deoxy-DCLHb in 0.1 M Hepes, pH 8, in the presence of 15 molar equivalents of STP was employed. The reactions were generally conducted as described in method 1. Experimental results are summarized in Supporting Information, Table S-4. RESULTS AND DISCUSSION

Polymerization of DCLHb with Polyamide CrossLinking Agents. All of the polyamide cross-linking agents employed in these studies were bifunctional

Bioconjugate Chem., Vol. 10, No. 6, 1999 1015

reagents. The activated bis(N-oxysuccinimidyl) diesters 1a and 1b can react with the -amino groups of lysine residues or the N-terminal amino groups of hemoglobin to form amide linkages between the polyamide and the protein. Maleimide derivatives, such as 3a and 3b, can react with lysine or cysteine residues of hemoglobin to form C-N and C-S linkages, respectively. 2a and 2b (that contain succinimidyl-N-oxycarbonyl end groups) can react with lysine residues or the N-terminal amino groups of hemoglobin to form urea linkages. The polyamide bis(aldehyde) 4a and 4b can polymerize DCLHb via reductive amination reactions, to yield DCLHb molecules linked by covalent C-N bonds. If both reactive end groups of a polyamide react with different DCLHb molecules, an intermolecularly crosslinked polyDCLHb is produced, and the SEC retention time is decreased. In contrast, if only one reactive terminus reacts with a DCLHb molecule and the other reactive terminus is deactivated (i.e., via hydrolysis), surface modification of DCLHb will result. As is true of a polyDCLHb, the SEC retention time of a surfacemodified DCLHb is decreased, but not as greatly as when DCLHb is polymerized. Historically, the size of hemoglobin derivatives has been assessed using size-exclusion chromatography (SEC). A series of protein standards having known molecular weights typically are used to calibrate the chromatographic conditions, so that retention times may be correlated with the molecular weight. A shortcoming of this relationship is that its rigor depends on the similarity of the test articles to the protein standards. The polyDCLHb derivatives we have evaluated have been modified using polyamides that both modify the surface and polymerize the protein. This type of modification renders them different from the protein standards. Therefore, the chromatographic retention time does reflect the relative size of the polyDCLHb molecules being separated, but it cannot be used to vigorously establish molecular weights. The targeted polymerized DCLHb product would be devoid of monomer (DCLHb) and high molecular mass polymers (defined as products having with molecular masses that exceed the exclusion limit of 1 × 106 Da a of TSK G4000SW column). With this objective in mind, the molecular mass of the polyamide, the polyamide reactive end groups, the DCLHb concentration, the number of molar equivalents of polyamide used (relative to DCLHb), and the reaction pH were varied. In addition to these reaction parameters, the effects of sodium tripolyphosphate (STP) on the polymerization of DCLHb were studied. In general, the methemoglobin content increased from 3 to 10% during the polymerization reaction. Methemoglobin is the oxidized form (Fe2+ to Fe3+) of hemoglobin and is nonfunctional, that is, does not transport oxygen. Therefore, to minimize the methemoglobin content of the polyDCLHb molecules, N-acetyl L-cysteine was added to the deoxygenated product. The reduction of methemoglobin was allowed to take place over a 12-24 h period prior to product workup. The effects of the DCLHb concentration on the course of polymerization were studied using 1a (MW 4200) (Table 1 and Figure 2). In general, polymerization of DCLHb with this polyamide gave a product mixture having a SEC profile consisting of three peaks. These peaks were identified as “monomer” (DCLHb and surfaced modified DCLHb), “oligomers” of DCLHb, and “high molecular weight (HMW) polymers” of DCLHb that eluted in the excluded volume of the column.


Hai et al.

Figure 3. Comparison of the SEC profiles of 1a (MW 4200) and 1a (MW 7600) polymerized DCLHb.

As expected, the data indicate that high DCLHb concentrations favor the formation of polymeric products, even when low molar ratios of the cross-linker to DCLHb were used. Thus, when the DCLHb concentration was 15 g/dL, the use of 3 molar equivalents of 1a (MW 4200) gave a product in which 60% of the DCLHb had been polymerized. In contrast, when the DCLHb concentration was 6 or 3 g/dL, the use of 3 molar equivalents of 1a (MW 4200) gave only 37 and 22% polymerized hemoglobin, respectively. The polymerization of DCLHb at higher DCLHb concentrations was difficult to control. The use of 2.5 molar equivalents of 1a (MW 4200) and a DCLHb concentration of 15 g/dL yielded a product mixture containing 2% of the undesired HMW polymers. The data are presented in Table 1. If the number of molar equivalents of the cross-linker was increased in an attempt to increase the extent of polymerization, the percentage of undesired

HMW polymers increased rapidly. However, if the DCLHb concentration was 3 g/dL (the lowest concentration that is practical for commercial manufacture), as many as 7 molar equivalents of 1a (MW 4200) could be used for polymerization. These conditions provided a product that was 50% polymerized and contained no HMW polymers. Moreover, the shift in retention times of the monomers indicated that the product was extensively surface modified. Surface modification of DCLHb may enhance the longevity in the systemic circulation and the biocompatibility of the polyDCLHb. Thus, the degree of polymerization and product composition could be controlled by altering the concentration of DCLHb and the molar ratio of the polyamide reagent to DCLHb. The molecular mass of the polyamide reagent played a key role in maximizing the efficiency of the polymerization reaction. DCLHb is a globular protein having a diameter of about 60 Å. Because of this relative bulki-



Table 1. Effect of DCLHb Concentration on the Polymerization Profile Obtained with 1a (MW 4200) SEC profile molar ratio of PA to DCLHb DCLHb 0

% monomer (RT)a

% oligomer

% HMW polymer

P50 (mmHg) (n)

100 (19.9)

0

0

32 (2.6)

[DCLHb] ) 15 g/dL, no STP 2.5 3 3.5 4

44 (18.1) 39 (17.3) 55 (17.4) gel

54 48 34

2 13 33

27 (1.7) 29 (1.6) 32 (1.7)

[DCLHb] ) 10 g/dL, no STP 1 1.5 2 2.5 3

100 (18.9) 54 (18.2) 41 (18.3) 33 (18.0) 23 (17.4)

0 46 58 60 50

0 0 1 7 27

30 (1.9) 33 (1.8) 30 (1.7) 31 (1.6) 36 (1.7)

[DCLHb] ) 6 g/dL, no STP 2 3 4 5 6

72 (18.3) 63 (17.5) 47 (16.9) 38 (16.5) 27 (16.0)

28 37 50 46 33

0 0 3 16 40

ND 38 (1.9) 42 (1.9) 42 (1.6) 44 (1.7)

[DCLHb] ) 3 g/dL, no STP 3 4 5 6 7 8 10

78 (17.3) 72 (17.0) 68 (16.6) 51 (16.0) 49 (15.6) 37 (15.9) 35 (15.4)

22 28 32 49 51 58 54

0 0 0 0 0 5 11

39 (1.9) 40 (1.8) 44 (1.9) 49 (1.8) 50 (1.7) 46 (1.5) 52 (1.6)

0 0 0 2 6 11

30 (1.9) 30 (1.8) 29 (1.8) 32 (1.7) 28 (1.6) 30 (1.7)

[DCLHb] ) 3 g/dL, 15 molar equivalents STP (relative to DCLHb) 3 64 (17.2) 36 4 49 (16.5) 51 5 41 (16.3) 59 6 36 (15.9) 62 7 36 (16.3) 58 8 30 (16.5) 59 a

Number in parentheses is the elution time in minutes of the corresponding peak maximum.

ness, a longer chain polyamide would be expected to facilitate intermolecular linking of DCLHb molecules; thus, the efficiency of polymerization was expected to increase with the chain length of the cross-linker. This was indeed the case. A comparison of the extent of DCLHb polymerization with 1a (MW 4200) and (the longer) 1a (MW 7600) shows that as much as 90% polymerization could be obtained with 1a (MW 7600). Moreover, no HMW polymers formed when the higher molecular weight (longer chain) 1a (MW 7600) was used (Table S-1). Thus, a shorter chain cross-linking agent is more useful for surface modification and polymerizing DCLHb, while a polyamide with a longer chain provides a higher degree of polymerization. Representative SEC profiles are shown in Figure 3. The polyamide cross-linking agents 1a and 1b contain reactive end groups that can be hydrolyzed; therefore, the reaction pH can affect the extent of polymerization. In a solution that contains DCLHb, the reagents 1a or 1b can react either with the amino groups on DCLHb to form amide bonds or can be hydrolyzed by water to carboxylic acids that can no longer react with DCLHb. Therefore, if one of the end groups of the polyamide reagent reacts with DCLHb via aminolysis and the end group under goes hydrolysis, surface modification of DCLHb will result. Thus, the reaction of a 1a or 1b derivative with DCLHb affords a mixture of polyDCLHb and surface-modified DCLHb molecules in which the

ratio of polymerized to surface-modified DCLHb molecules is dependent on the relative rates of aminolysis and hydrolysis of the activated end groups of the polyamides. Because the nucleophilicity of the amino groups of DCLHb depends on their lack of protonation, the rate of aminolysis will depend on the buffer pH. The effect of the buffer pH on the degree of polymerization was evaluated by performing DCLHb polymerizations at pH 8 (0.1 M Hepes) and pH 9 (0.1 M borate) and comparing the product profiles. The product distribution of the polymerization of deoxyDCLHb (3 g/dL) at 5 °C in 0.1 M Hepes, pH 8, or 0.1 M borate, pH 9, containing 15 molar equivalents of STP were compared using both 1b (MW 5200) and 1a (MW 5600). The data presented in Table S-2 illustrate several points. First, using the same Polyamide/DCLHb molar ratio, reactions performed at pH 9 gave polyDCLHb containing less surface-modified DCLHb (monomer) and more oligomers than those performed at pH 8. For example, at pH 8, polymerization of DCLHb (3 g/dL) with 3.5 molar equivalents of 1b yielded a product mixture containing 8% surface modified DCLHb and 90% oligomers, about 1/4 of which were the higher molecular weight Oligomer II fraction, defined as molecules with peak maxima having retention times of 11.0-14.7 min. At pH 9, oligomerization was almost quantitative; the corresponding product mixture contained at most traces of surface-modified DCLHb, and approximately a third was the Oligomer II fraction.


Similar results were obtained with 1a (MW 5600). For example, with a 1a/DCLHb molar ratio of 3, at pH 8, polymerization with 1a (MW 5600) yielded a product mixture containing 10% surface-modified DCLHb and 90% oligomers, about 1/3 of which were the higher molecular weight Oligomer II fraction. In contrast, at pH 9, the corresponding product mixture contained 5% surface-modified DCLHb and 95% oligomers, approximately half of which are the Oligomer II fraction. Second, similar polyDCLHb profiles could be obtained by using lower polyamide/DCLHb molar ratios if the reaction was performed at pH 9 rather than at pH 8. For example, the polyDCLHb obtained from 1b (MW5200) at pH 9 using a 3:1 molar ratio of 1b/DCLHb had a similar SEC profile to that obtained at pH 8 using a 3.5:1 molar ratio of 1b/DCLHb. Finally, the P50 and n values of Poly-DCLHb derived from 1b were not extensively affected by the reaction pH. The effect that competitive hydrolysis of a reactive end group of a polyamide has on the extent of polymerization was demonstrated by the comparison of the polymerization of DCLHb with 1a (MW 4200) (a cross-linking agent subject to hydrolysis) with the polymerization observed using 3a (MW 4080), a maleimide-terminated crosslinker of comparable length that will not hydrolyze under the reaction conditions. The reactions of the two crosslinkers were similar in two respects: (a) the yield of the oligomer increased with the number of molar equivalents of cross-linker used and (b) the elution times of the monomer decreased with the number of molar equivalents of cross-linker used, indicating that surface modification of DCLHb had occurred. The data are presented in Tables S-2 and S-3. The reactions of these two cross-linkers also differed in two other respects. First, the RP-HPLC profiles (Figure 4) suggest that 3a (MW 4080) reacted specifically with DCLHb. A new peak, the β1 peak, was detected at all ratios of 3a (MW 4080) tested. Maleimide reagents, such as 3a, are expected to bind specifically to cysteine-β 93 residues, and this modification would shift the retention time of the β-subunit. Specific reaction of the β-chain was also supported by the decrease in titratable thiol residues (Figure 5); about 65 and 90% of the titratable thiol groups are modified when 1 and 2 equiv of 3a (MW 4080) are used, respectively. Second, thiol modification by 3a (MW 4080) resulted in a decrease in the P50 values of the polymerized products to 18-20 mmHg, whereas modification by 1a did not effect the P50 values under similar reaction conditions. A decrease in the P50 value is an expected consequence of cysteine-β 93 modification (14, 15). Finally, the effectiveness of two other types of polyamide cross-linking agents was assessed. DCLHb was polymerized with 4a (MW 4000), a polyamide containing aldehyde end groups, via reductive amination. The reductive amination reactions were performed in 0.1 M Hepes, pH 7.0, in the presence of either sodium cyanoborohydride, borane-pyridine, sodium borohydride, or borane-dimethylamine. The data are presented in Table S-3. Unexpectedly, we found that reduction of the imine with borane-dimethylamine or sodium borohydride did not produce polyDCLHb, although other investigators have reported that these are useful reducing agents for reductive amination reactions (16). In contrast, when either sodium cyanoborohydride or borane-pyridine was used, 4a (MW4000) provided both surface-modified and polymerized DCLHb, although the efficiency of the latter was modest. Under the best conditions found, the polymerized products still contain 38-48% of modified

Hai et al.

Figure 4. Reversed-phase HPLC profiles of 3a (MW 4800) polymerized DCLHb.

Figure 5. Relationship between the number of free thiols and the P50 value of 3a polymerized DCLHb.

monomer. Reduction with borane-pyridine produced a polyDCLHb having values of P50 and n in the range 3336 mmHg and 1.9-2.1, respectively. Thus, the polymerization of DCLHb by reductive amination maintained the P50 value and cooperativity of DCLHb. DCLHb was also polymerized with 2a (MW 2800) and 2a (MW 5600), polyamides having succinimidyl-N-oxy-


Figure 6. Relationship between the P50 value of polymerized DCLHb and the concentration of DCLHb.

Figure 7. Relationship between the P50 value and the retention time of the monomer peak of 1a (MW 4200) polymerized DCLHb.

carbonyl (activated urethane) termini. In each case, a 3 g/dL deoxyDCLHb solution in 0.1 M Hepes, pH 8, was allowed to react at 5 °C. Six molar equivalents of 2a (MW 2800) and 4 molar equivalents of the longer 2a (MW 5600) were used in the respective polymerizations. Both polyDCLHbs had acceptable SEC profiles and appeared to be suitable for additional biological screening. However, the SEC profiles changed during refrigerated storage of the products, suggesting that the resulting urea linkages were unstable. Controlling the Oxygen-Binding Characteristics of the PolyDCLHbs. Three means for controlling the oxygen-binding characteristics of the polyDCLHb products were studied. First, the polymerization reaction was performed in the presence of STP. Under these conditions, the P50 of the product was maintained in the range 27-36 mmHg, the same range as that of DCLHb. However in the absence of STP, the P50 of the product was dependent upon the starting concentration of DCLHb used in the polymerization and the molar equivalents of the polyamide reagent used. The data are presented in Figure 6. For each concentration of DCLHb used, the P50 increased with an increase in the molar equivalents of the polyamide used in the reaction. The highest P50 values were obtained with dilute starting concentrations of DCLHb. It was also noted, as the graph in Figure 7 illustrates, that there is an approximately linear relationship between the P50 value of the polyDCLHb solution and the SEC retention time of the monomer. The data suggest that an increase in the extent of surface modi-


fication of DCLHb correlates with an increase in the P50 value of the polymerized product. A comparison of the SEC profiles of the polymerized products indicates that STP has no detectable effect on the product composition despite the effect on the P50 value. Since STP is known to bind to hemoglobin at the 2,3-diphosphoglycerate (DPG) binding site (17, 18), one reasonable interpretation of this observation is that the presence of STP in the DPG-binding site protects the amino acid residues in that region from modification by 1a (MW 4200). Altering the reactive end group provided a second means for controlling the oxygen-binding characteristics of the polyDCLHb products. A P50 value of about 20 mmHg and a decrease of the cooperativity value to 1.7 (n40-60) were obtained by polymerization with a polyamide containing a maleimide end group such as 3a (MW 4080). As shown in Figure 5, the alkylation of the thiols of cys β93 has a profound effect on the oxygen binding characteristics of the polymerized products. Last, the oxygen-binding characteristics of the poly-DCLHb products could be modified by polymerization of either oxyDCLHb or deoxyDCLHb. Other investigators have shown that polymerization in the T-conformation of hemoglobin (deoxyHb) or in the R-conformation (oxyHb) yields polymerized hemoglobins that are partially locked in the T- or R-conformation, respectively (2). The oxygen affinity of a polyHb prepared from a T-state Hb is expected to be different from that prepared from a R-state Hb. To demonstrate that this also applied to the polyamide-polymerized DCLHb, deoxyDCLHb (3 g/dL or 6 g/dL) or oxyDCLHb (3 g/dL or 6 g/dL) in 0.1 M Hepes, pH 8, was polymerized with 1a (MW 5600) in the presence of 15 molar equivalents of STP at 5 °C. In general, SEC profiles of the polyDCLHbs produced under these conditions gave four SEC bands: monomer band, oligomer I band (defined as molecules having peak maxima with retention times of 15.2-17.2 min), oligomer II band (defined as molecules having peak maxima with retention times of 11.0-14.7 min), and HMW polymer band (defined as molecules having molecular sizes exceeding the exclusion limit of the columns) (see Table S-4). When oxyDCLHb was polymerized with 1a (MW 5600), the polyDCLHb products had P50 and n values in the range of 18-27 mmHg and 1.5-2.3 (n40-60), respectively. In contrast, when deoxyDCLHb was polymerized, these values were in the range of 23-36 mmHg and 1.82.5(n40-60), respectively. In summary, we have demonstrated the ability to control the polymerization of DCLHb and the oxygenbinding properties of the polyDCLHb product. This was accomplished by either altering the polyamide crosslinking reagent, the reaction pH, the DCLHb concentration, the molar equivalents of the polyamide reagent, or by conducting the polymerization with either oxy- or deoxyDCLHb. Supporting Information Available: Data describing the polymerized DCLHb products are presented in Tables S-1-S-4. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Bolin, R., and DeVenuto, F. (1983) Hemoglobin solution as a blood substitute. Adv. Blood Res. 1-7. (2) Labrude, P., Chaillot, B., Dellacherie, E., Stoltz, J. F., and Vigneron, C. (1986) Hemoglobin solutions as oxygen carriers: Ligands and other molecules interactions with hemoglobin. Adv. Biosci. 54, 161-172.

1020 Bioconjugate Chem., Vol. 10, No. 6, 1999 (3) Leonard, M., Neel, J., and Dellacherie, E. (1984) Synthesis of monomethoxypolyethylene-bovine hemoglobins. Tetrahedron 40, 1581-1584. (4) Sehgal, L. R., Rosen, A. L., Gould, S. A., Sehgal, H. L., and Moss, G. S. (1983) Preparation and in vitro characteristics of polymerized pyridoxylated hemoglobin. Transfusion 23, 158162. (5) Iwasaki, K., Iwashita, Y., and Okami, T. (1987) Hemoglobin combined with a poly(alkylene oxide), U.S. Patent 4,670,417. (6) Tam, S. C, Blumenstein, J., and Wong, T. F. (1976) Soluble dextran-hemoglobin complex as potential blood substitute. Proc. Natl. Acad. Sci. U.S.A. 73, 2128-2131. (7) Chang, J. E., and Wong, T. F. (1977) Soluble dextranhemoglobin complexes of different molecular sizes. Can J. Chem. 55, 398-403. (8) Hai, T. T., Pereira, D. E., and Nelson, D. J. (1998) Synthesis of water soluble, non immunogenic polyamide cross-linking agents. Bioconjugate Chem. 9, 645-654. (9) Hai, T. T., and Nelson, D. J. (1994) Water soluble, non immunogenic polyamide cross-linking agents. PTC Int. Appl. WO 94/12220. (10) Hai, T. T., Nelson, D. J., and Srnak, A. (1992) Process for the production of cross-linked hemoglobin in the presence of sodium tripolyphosphate, U.S. Patent 5,128,452. (11) Chatterjee, R., Welty, E. V., Walder, R. Y., Pruitt, S. L., Roger, P. H., Arnone, A., and Walder, J. A. (1986) Isolation and characterization of a new hemoglobin derivative cross-

Hai et al. linked between the R chains (Lysine 99R1-Lysine 99R2). J. Biol. Chem. 261, 9929-9937. (12) Van Assendelft, O. W., and Zijlstra, W. G. (1975) Extinction coefficients for use in equations for the spectrophotometric analysis of hemoglobin mixtures. Anal. Biochem. 69, 43-48. (13) Neis, J. M., Van Gemert, P. J. L., Roelofs, H. M. J., and Henderson, P. T. H. (1984) Disappearance of free SH-groups in hemoglobin of man, rat and rabbit after exposure to alkylating agents in vitro. Toxicology 31, 319-327. (14) Riggs, A. (1961) The binding of N-Ethylmaleimide by human hemoglobin and its effect upon the oxygen equilibrium. J. Biol. Chem. 236, 1948-1954. (15) Garel, M. G., Beuzard, Y., Domenget, C., Martin, J., Galacteros, F., and Rosa, J. (1982) Binding of 21 thiol reagents to human hemoglobin in solution and intact cells. Eur. J. Biochem. 123, 513-519. (16) Geoghegan, K. F., Cabacungan, J. C., Dixon, H. B. F., Feeney, R. E. (1981) Alternative reducing agents for reductive methylation of amino groups in proteins. Int. J. Pept. Protein Res. 17, 345-352. (17) Chanutin, A., and Herman, E. (1969) The interaction of organic and inorganic phosphates with hemoglobin. Arch. Biochem. Biophys. 131, 180-184. (18) Chanutin, A., and Curnish, R. R. (1967) Effect of organic and inorganic phosphates of the oxygen equilibrium of human erythrocytes. Arch. Biochem. Biophys. 121, 96-102.

BC9900486

Polymerization of Diaspirin Cross-Linked Hemoglobin (DCLHb) with

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