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Chemical characterization of pyridoxalated hemoglobin polyoxyethylene conjugate
Biochimica et Biophysica Acta 1476 (2000) 53^65 www.elsevier.com/locate/bba
Chemical characterization of pyridoxalated hemoglobin polyoxyethylene conjugate Todd L. Talarico *, Katherine J. Guise, Cyrus J. Stacey Apex Bioscience Inc., P.O. Box 12847, Research Triangle Park, NC 27709, USA Received 2 July 1999; received in revised form 14 September 1999; accepted 16 September 1999
Abstract Pyridoxalated hemoglobin polyoxyethylene conjugate (PHP) was developed in the 1980s as an oxygen carrier and is now under development for treatment of nitric oxide-dependent, volume refractory shock. PHP is made by derivatizing human stroma-free hemoglobin with pyridoxal-5-phosphate and polyoxyethylene (POE). A unique aspect of using POE for modification is that unlike its mono-methoxy polyethylene glycol (PEG) relatives, POE is bifunctional. The result of derivatization of stroma-free hemoglobin is a complex mixture of modified hemoglobin and other red cell proteins. The molecular weight profile, based on size exclusion chromatography, is bimodal and has a number average molecular weight of approximately 105 000 and a weight average molecular weight of approximately 187 000. The mixture of hemoglobin molecules has on average 3.3 pyridoxal and 5.0 polyoxyethylene units per tetramer. A portion of the tetramers are linked by POE crosslinks. The hemoglobin tetramers retain their ability to dissociate into dimer pairs and only a small percentage of the dimer pairs are not modified with POE. The SDS-PAGE profile exhibits the ladder-like appearance commonly associated with polyethylene glycol-modified proteins. The isoelectric focusing profile is broad, demonstrating a pI range of 5.0^6.5. The hydrodynamic size of PHP was determined to be approximately 7.2 nm by dynamic light scattering. Soluble red blood cell proteins, such as catalase, superoxide dismutase, and carbonic anhydrase, are present in PHP and are also modified by POE. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Hemoglobin; Polyethylene glycol; Polyoxyethylene; Blood substitute; Shock
1. Introduction Hemoglobin accounts for greater than 90% of soluble protein in a red blood cell and is responsible for the transport of oxygen from the lungs to other tissues in the body [1]. Hemoglobin Ao and its variants from humans and hemoglobins from a large number of organisms have been studied extensively [2^5]. The crystal structures of a number of di¡erent
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hemoglobins have been determined and their function in transport of oxygen, carbon dioxide and nitric oxide has been elucidated [2,6,7]. Inside red blood cells, human hemoglobin Ao exists as a tetramer containing 2 K and 2 L subunits. Stable dimers consisting of an K and L subunit are in equilibrium with the tetramer form in solution and the dissociation state of the tetramers depends on solution characteristics, such as hemoglobin concentration, salt content and pH. Each of the subunits contains an iron protoporphyrin which must exist in the ferrous state in order for hemoglobin to transport oxygen [8]. During the course of routine functioning in the
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body, hemoglobin is exposed to a number of insults which result in the oxidation of the ferrous form of the iron to the ferric form. These insults include exposure to hydrogen peroxide, peroxynitrite and nitric oxide [9^11]. These insults are opposed by antioxidant enzymes and reductases [12,13], which e¤ciently maintain more than 99% of the red cell hemoglobin in the ferrous state [14]. A number of hemoglobin-based therapeutics are currently under development by companies in the United States and abroad [15^18]. Hemoglobin therapeutics have been designed as oxygen-carrying £uids useful for blood replacement during surgery and in trauma, enhancers of radiation therapy, and scavengers of nitric oxide [19,20]. Hemoglobin, once removed from the red cell, cannot be used for these therapeutic indications without modi¢cation due to renal toxicity, as described by Savitsky et al. [21]. In addition, the plasma half-life of unmodi¢ed hemoglobin is short and the oxygen a¤nity of hemoglobin in plasma may be too great to allow e¤cient delivery of oxygen in those applications where oxygen delivery is desired [22]. These limitations to the use of unmodi¢ed human hemoglobin have been circumvented by chemical modi¢cation of the hemoglobin in one or more of the following ways: (1) crosslinking K, L dimers; (2) polymerizing hemoglobin; or (3) binding polymers to the surface of hemoglobin [23^ 25]. One such product, pyridoxalated hemoglobin polyoxyethylene conjugate (PHP) was initially developed in the 1980s as an oxygen-carrying solution [26]. Currently, PHP is in clinical trials for treatment of nitric oxide-dependent, volume refractory shock [27,28]. PHP is produced from erythrocyte lysate made from outdated human red blood cells. The method used provides for separation of the hemoglobin from red cell stroma and potential adventitious agents, but not from many of the enzymes naturally associated with hemoglobin in red cells. The hemoglobin is pyridoxalated to reduce the oxygen a¤nity [29]. The pyridoxalated stroma-free hemoglobin is then modi¢ed with the homo-bifunctional molecule POE to increase the hydrodynamic volume (apparent molecular weight) of the molecule. The resulting modi¢ed hemoglobin entity is puri¢ed to remove residual reactants, formulated in electrolytes, and rendered sterile for use as a parenteral drug.
This conjugated hemoglobin has unique characteristics conferred by the decoration with POE. Currently, there are a number of proteins conjugated with PEG which have been approved by the Food and Drug Administration for human use [30]. The chemical characterization of many PEG-conjugated proteins has been described [30^33], but there are few examples of characterization of POE-conjugated proteins. Among these, Sato et al. reported on the characterization of superoxide dismutase-POE-deferoxamine and Iwashita performed some preliminary characterization of POE-modi¢ed hemoglobin (PHP)[26,34,35]. Since PHP will be used as a large volume parenteral drug in a population which is typically critically ill, understanding the safety and biochemical nature of the product is important. This report describes the chemical characterization of PHP. The characterization provides new information about the nature of the chemical modi¢cation and the association of the subunits of the molecule as they exist in solution. The association of subunits with other proteins also occurs due to the unique properties of the modi¢cation agent used during production of PHP. 2. Materials and methods PHP was produced by the Apex Bioscience Manufacturing group based upon the synthesis method described by Iwashita and colleagues [35]. The production process was modi¢ed as described by Talarico et al. [36]. All PHP used in this study met the requirements for release by the Apex Quality Control group and were representative of material used in clinical trials. The K,K-crosslinked hemoglobin was a gift from the Walter Reed Army Institute of Research. In addition to PHP, several hemoglobin derivatives were synthesized and analyzed. Pyridoxalated hemoglobin was synthesized by reaction of excess pyridoxal-5-phosphate [29] with deoxygenated stroma-free hemoglobin solution and was reduced with sodium borohydride (Alfa Aesar, Ward Hill, MA). PEG hemoglobin was synthesized using monomethoxy polyethylene glycol (MW 3000) activated with N-hydroxysuccinimide (Shearwater Polymers, Huntsville, AL). Various molar excesses of PEG to pyri-
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doxalated hemoglobin were reacted in bu¡er containing 50 mM sodium phosphate and sodium chloride, pH 8.0 for 1 h at room temperature. The reaction mix was dialyzed at 4³C for 24 h against phosphate bu¡er to remove residual reactants. Crosslinks speci¢c to L-93 cysteine were made on pyridoxalated hemoglobin with a bifunctional maleimide PEG, MW 3400 (Shearwater Polymers). The reaction was performed with a 1.5-fold molar excess of crosslinker to hemoglobin at room temperature for 3 h. The reaction mix was dialyzed against phosphate bu¡er to remove residual reactants. Size exclusion chromatography (SEC) for molecular weight determination was performed on a Waters Alliance 2690 separations module with detection at A280 (Waters, Milford, MA). All data were analyzed with Waters Millenium software equipped with the GPC processing package. The separation was performed at 30³C on a BioSEP-S-4000 column (Phenomenex, Torrance, CA) using a 0.1 M potassium phosphate mobile phase, pH 6.8, containing 13% ethanol. Ten microliters of a 30 mg/ml solution of PHP was injected and the molecular weight of the sample was determined from a standard curve derived from the retention times of protein reference standards (Bio-Rad, Hercules, CA). SEC for estimation of the percentage of unmodi¢ed dimer was performed on a 410 series bio LC pump (Perkin-Elmer, Norwich, CT) equipped with a Rheodyne injector and a Waters 990 photodiode array detector at A280 . All data were analyzed with the software package supplied with the detector. The separation was accomplished with 20 Wl of a 5 mg/ml sample on a SuperDex 75 column (Pharmacia Biotech, Piscataway, NJ) using a 25 mM Tris HCl pH 8.0/1.0 M magnesium chloride mobile phase at a £ow rate of 0.5 ml/min. Hemoglobin and methemoglobin concentrations were determined using an IL 482 CO-Oximeter (Instrumentation Laboratory, Lexington, MA). The extent of pyridoxalation was determined following digestion of PHP to liberate the phosphate. PHP was dialyzed against USP puri¢ed water and 25 mg of PHP was placed in a glass test tube. Digest solution composed of sodium molybdate, sulfuric acid and perchloric acid was added to the tube and the mix was heated at 180³C for 60 min. The phosphate content was then determined using a kit for
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determination of inorganic phosphate according to the manufacturers instructions (Sigma, St. Louis, MO, Procedure 670). The determination of the extent of modi¢cation of the hemoglobin with POE was performed after digestion of the PHP with 6 N HCl for 6 h at 110³C to liberate the POE. The POE content of the digest solution was determined by the method of Skoog [37] by comparison to POE standards (Nippon Oil and Fats, Tokyo, Japan). Titrisol reagent was obtained from Merck (Germany). Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of PHP and other hemoglobin species was performed on 10% NuPAGE gels (Novex, San Diego, CA) under reducing conditions. The separation was developed with 3-(N-morpholino)ethane sulfonic acid-SDS running bu¡er as described by the manufacturer on the gel insert instructions. Molecular weight standards (Mark 12, Novex) were included on each gel. Proteins were visualized by staining with colloidal blue Coomassie G-250 (Novex) according to the manufacturer's instructions. Quantitation of protein bands was performed by scanning the gel using a pdi 420oe scanner (BioRad) and analyzing each lane with Quantity One software. Western blots were performed using antibodies to the following human enzymes: Cu/Zn superoxide dismutase (SOD), catalase, and carbonic anhydrase II (The Binding Site, Birmingham, UK). SDS-PAGE separation was achieved on 10% NuPAGE gels as described above. Depending on the sensitivity of the antibody, PHP was loaded onto the gel at 10^ 50 Wg hemoglobin. Human erythrocyte enzymes corresponding to each antibody were obtained from Sigma (St. Louis, MO) to serve as positive controls. The load of each enzyme was based upon the antibody sensitivity, and ranged from 10 to 50 ng. A prestained molecular weight marker (SeeBlue, Novex) was also included with each blot to assess transfer e¤ciency. The proteins were blotted onto nitrocellulose membranes (Novex) in Tris-Glycine transfer bu¡er. Upon completion of the transfer, the membranes were blocked in 3% milk in 1UTris bu¡ered saline (TBS) for 1.5^2 h. The primary antibodies were diluted 1:1000 in 1% milk in 1UTBS (50 Wl in 50 ml) with or without 0.025% Tween-20 (BioRad) and incubated overnight at 42³C. The mem-
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branes were washed in 1UTBS with 0.025% Tween20 then placed in secondary antibody. The membranes were incubated in peroxidase conjugated donkey anti-sheep/goat IgG (HpL) (The Binding Site), diluted in the same manner as the primary antibody, for approximately 3 h at 42³C, followed by another set of TBS/Tween-20 washes. Visualization of catalase and carbonic anhydrase was accomplished with tetramethyl benzidine (TMB) substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD). The SOD blot was developed in SuperSignal Chemiluminescent substrate (Pierce, Rockford, IL). The blot was exposed to Kodak Biomax ¢lm (Rochester, NY) for 10 s and processed using a Konica (Japan) QX-70 processor. IEF analysis was performed on 3^9 IEF gels according to the manufacturer's directions (Novex). IEF 3^9 standards were used for calibration (BioRad). Gels were stained with Coomassie blue and quantitated as described for the SDS-PAGE analysis. Hydrodynamic size measurements were performed on a DynaPro Molecular Sizing Instrument (Protein Solutions, Charlottesville, VA). PHP samples were diluted to approximately 11 mg/ml in 100 mM sodium phosphate, pH 7.2. Hemoglobin samples were diluted to approximately 6 mg/ml in the same bu¡er. All samples were ¢ltered prior to injection with a 100-nm Whatman Anodisc Membrane (Whatman, Clifton, NJ) for particulate removal. Samples were analyzed at 20³C, and scattering signals were collected at 90³ from the incident laser beam. Hydrodynamic size distributions were generated from the resulting autocorrelation function with DynaLS Non-negative least squares algorithm (Copyright 1996, 97 Alexander Goldin, Nickolay Sidorenko). The result provided the mean peak size and standard deviation for each peak resolved. Catalase and derivatized catalase in PHP were puri¢ed by a¤nity chromatography. A¤nity resin was prepared by attachment of anti-human erythrocyte catalase antibody (The Binding Site) to CNBr Sepharose CL-4B (Pharmacia Biotech) as recommended by the manufacturer. A 200-mg PHP sample in 0.1 M Tris-HCl, pH 8.0/0.5 M NaCl was passed through a 1-ml column of the resin. Bound material was eluted in 200 mM glycine HCl and immediately neutralized with 1 M Tris HCl, pH 8.0. The elution was performed with a peristaltic pump (Master£ex, Cole-
Parmer, Chicago, IL), monitored at A280 with a UV detector (Pharmacia Biotech) and recorded on a strip chart recorder. The collected fractions were separated by SDS-PAGE, blotted onto nitrocellulose, and protein species were detected with antibody to hemoglobin (Chemicon, Temecula, CA) or catalase as described above. MALDI mass spectrometry (MS) analysis was performed on PHP which had been fractionated by SDS-PAGE and then blotted onto nitrocellulose. For analysis, PHP was separated on a 10% Tris-glycine gel (Novex), blotted onto nitrocellulose, and stained with Ponceau S to visualize the protein bands. Slices of the membrane corresponding to distinct protein bands were cut from the blot using a razor blade and were dissolved in acetone for application to the MALDI target [38]. The MALDI investigations were performed using a Bruker Pro£ex+ (Bruker Daltronics, Billerica, MA) linear MALDI-TOF instrument with a 1.2-m £ight tube equipped with a nitrogen laser. Detection was performed with a dual microchannel plate detector at a typical detector voltage of 1.55 V. A sampling rate of 1 GHz and pulsed ion extraction were used during data collection. The data were collected and processed with the Xacq 3.0 and Xmass 3.0 software packages (Bruker). 3. Results and discussion The molecular weight (MW) of PHP was estimated by SEC based upon retention time as compared to protein standards. Nine manufacturing lots of PHP were analyzed and the peak average MW was determined to be 106 164 þ 3385, the number average MW was determined to be 105 122 þ 3566 and the weight average MW was determined to be 187 000 þ 8300. The pro¢le of the chromatogram indicated that the MW distribution was bimodal as shown in Fig. 1. The polydispersity of the PHP was 1.778 þ 0.031. Typical peak molecular weights from the two main species of the bimodal distribution were 106 000 and 300 000. The relative peak areas were typically on the order of 70% (low MW component) and 30% (high MW component) when approximated using a drop baseline for integration. Stroma-free hemoglobin had a peak MW of 38 214 and hemoglobin crosslinked
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Fig. 1. SEC chromatogram of PHP and unmodi¢ed hemoglobin. PHP displays a bimodal distribution. Unmodi¢ed hemoglobin elutes after the majority of PHP.
between the K subunits had a peak MW of 39 288 when analyzed on the same chromatography system. Derivatization of hemoglobin to produce PHP includes attachment of POE and pyridoxal-5-phosphate. POE was released from PHP by digestion with hydrochloric acid and quantitated spectrophotometrically after it was complexed with barium/iodine. The analysis of ¢ve lots of PHP demonstrated that on average 5.0 þ 0.4 POE molecules were attached to each hemoglobin tetramer. This number agreed with the estimated extent of PHP derivatization based upon chromatographic analyses performed for process POE mass balances [39]. However, Iwashita et al. reported the presence of approximately 6 mol of POE per mol of hemoglobin in a previous study [26]. Future studies to examine the POE content of PHP using a chromatographic quantitation technique may resolve the reported differences [39]. The number of pyridoxal-5-phosphate molecules attached was determined by digestion of PHP followed by assay for inorganic phosphate con-
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tent. Each hemoglobin tetramer was found to contain 3.3 þ 0.1 pyridoxal-5-phosphate molecules based upon the analysis of ¢ve lots of PHP. This number was in agreement with a previous report by Iwashita et al. [26]. The relative percentages of POE-modi¢ed to unmodi¢ed K^L dimer pairs was determined by SEC performed under dissociating conditions where the SEC mobile phase contained 1 M magnesium chloride to promote dimer dissociation. The position of the unmodi¢ed dimer peak was con¢rmed by co-elution with puri¢ed hemoglobin and by performing studies in which puri¢ed hemoglobin was added to PHP samples. The percentage of unmodi¢ed dimer pairs in nine lots of PHP was determined to be 6.6 þ 1.3% with a range from 4.9 to 8.6%. The ability of PHP to separate into dimer pairs illustrates one di¡erence between this class of modi¢ed hemoglobins and those that are crosslinked between dimer pairs (such as K,K-crosslinked hemoglobin). PHP retains the ability to form an equilibrium mix of species in solution because its dimer pairs are free to dissociate. This equilibration between di¡erent species was demonstrated by addition of unmodi¢ed hemoglobin to PHP. Fig. 2 contains SEC chromatograms for PHP performed under nondissociating conditions (as was shown in Fig. 1). Fig. 2A represents SEC chromatograms on a series of samples in which puri¢ed hemoglobin was added to PHP to make a ¢nal concentration of 0, 5, 10 and 20% with respect to the puri¢ed hemoglobin. Additions of hemoglobin to PHP were performed such that the total hemoglobin concentration in samples and amount of hemoglobin loaded on the SEC column were identical in all analyses. The series of chromatograms show a progressive decrease in the two main peak heights and a progressive increase in the region of lower molecular weight hemoglobin. As a result of the additions, the overall retention time of all the PHP species shifted to longer and longer retention times. The PHP sample spiked with 20% hemoglobin possessed a distinct shoulder which co-migrated with puri¢ed hemoglobin (see Fig. 2C). Additions of hemoglobin to 5 or 10% did not result in formation of a distinct shoulder on the chromatograms but resulted in the broadening of the molecular weight distribution. The broadening can be noted by following the migration of the back-end
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In contrast to the chromatograms generated after addition of unmodi¢ed hemoglobin, the addition of K,K-crosslinked hemoglobin to PHP resulted in the chromatograms shown in Fig. 2B. In this case, dimer pairs of the K,K-crosslinked hemoglobin could not dissociate and therefore could not recombine with PHP. Additions of crosslinked hemoglobin into PHP solution to ¢nal concentrations of 0, 5, 10 and 20% resulted in a peak which co-eluted with K,K-crosslinked hemoglobin injected separately (Fig. 2C). The series of chromatograms show a progressive decrease in the two main peak heights and a progressive increase in the peak corresponding to K,K-crosslinked hemoglobin. The addition of crosslinked hemoglobin to PHP did not increase the peak retention time of the two main PHP peaks in the chromatogram. This indicated that the PHP and K,K-crosslinked hemoglobin did not undergo subunit exchange. Based upon the chromatograms generated by ad-
Fig. 2. SEC chromatograms of PHP spiked with hemoglobin. (A) Chromatograms representing PHP spiked with unmodi¢ed hemoglobin at ¢nal contents of 0, 5, 10 and 20% of the total hemoglobin concentration. The changes observed in the chromatograms proceed in order corresponding to the unmodi¢ed hemoglobin additions from 0 to 20% as shown. (B) Chromatograms representing PHP spiked with K,K-crosslinked hemoglobin at ¢nal contents of 0, 5, 10 and 20% of the total hemoglobin concentration. (C) Chromatograms representing unmodi¢ed hemoglobin and K,K-crosslinked hemoglobin analyzed in the absence of PHP. For the labeling on the chromatograms 0U is 0% and 20U is 20%.
of the chromatogram in Fig. 2A as higher levels of unmodi¢ed hemoglobin are added to PHP. This observation is consistent with dissociation and re-arrangement between dimers in PHP and puri¢ed hemoglobin; unmodi¢ed dimers exchanged with the PHP dimers would result in new species of PHP molecules with lower molecular weights.
Fig. 3. SDS-PAGE analysis of PHP on 10% gels. PHP exhibits the ladder pattern typical of PEG modi¢ed proteins. The fastest migrating band corresponds to the migration of K and L subunits which do not contain POE (band 1). Bands 2 and 3 correspond to subunits with 1 or 2 POE units. Band 4 is a tight band that has not been conclusively identi¢ed. Bands 5 and greater are apparent either as major individual species or as a smear at the most slowly migrating positions.
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dition of hemoglobin solutions to PHP, it should be possible to predict the limit of detection of unmodi¢ed hemoglobin in PHP solutions using the SEC technique. If it was assumed that unmodi¢ed dimers combined with modi¢ed and unmodi¢ed subunits with equal preference and there are approximately 6.6% unmodi¢ed dimers present in PHP solutions, one would expect 0.066U0.066 occurrences of unmodi¢ed tetramer. Such a scenario would lead to approximately 0.4% unmodi¢ed tetramer in a typical PHP lot. Based upon the spiking experiments performed with PHP, unmodi¢ed tetramers present at 0.4% would not be detectable in PHP solutions. Even additions of unmodi¢ed hemoglobin to 20% of the total hemoglobin content in PHP solutions would only be expected to yield approximately 4% unmodi¢ed tetramer when dimers were free to recombine with modi¢ed hemoglobin. Interestingly, a 20% spike of unmodi¢ed hemoglobin can be detected as a distinct shoulder on the PHP peak, similar to a 5% spike of K,K-crosslinked hemoglobin. The modi¢cation of hemoglobin at the K and L subunit level was visualized by performing SDSPAGE analysis and staining with Coomassie blue. A typical gel is shown in Fig. 3 and the bands corresponding to di¡erent protein species are numbered. The pro¢le of the material appeared ladder-like where K and L subunits which were not modi¢ed with POE migrated with approximately the same mobility as the protein hen egg white lysozyme (MW = 14 400). The heterogeneity of the low molecular weight species was due to the pyridoxalation of the hemoglobin subunits (see Fig. 4, lanes 6 and 8). Similar ladder-like pattern of migration of the higher MW bands of PHP has been observed for a number of PEG derivatized proteins [40,41]. Table 1 MALDI-TOF analysis of hemoglobin and PHP separated by SDS-PAGE Sample
Band number
Molecular weight
Hemoglobin
1
PHP
1
PHP PHP PHP PHP
2 3 4 5
15324 16090 15422 16125 18978 22957 23809 18222
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The protein bands which migrated with a MW of 21 500 and larger protein standards were assumed to represent hemoglobin subunits to which POE was bound. This was con¢rmed by MALDI mass spectrometry analysis of PHP separated by SDS-PAGE and blotted onto nitrocellulose. Individual bands were cut from the nitrocellulose, dissolved in acetone, and their MW was determined by MALDI mass spectrometry. Samples of puri¢ed human hemoglobin were included as a standard for molecular weight analysis by mass spectrometry. Puri¢ed hemoglobin samples separated by this method contained a single band which co-migrated with the 14 400 MW protein standard. Separation of the K and L subunits was not observed on the 10% gels used for these experiments; however, MALDI-MS analysis of the band resolved two species with molecular weights of 15 324 and 16 090 (Table 1). These MWs corresponded to the expected values for K and L subunits, respectively. In the PHP samples, band 1 contained proteins with molecular weights of 15 422 and 16 125. Pyridoxal-5-phosphate has a MW of 247 and these molecular weights probably re£ect a mixture of pyridoxalated and unadulterated subunits. The second band on the gel (MW = 18 978) was considered to be representative of mixture of K and L subunits, some pyridoxalated, containing one POE molecule (MW = approximately 3100). Band 3 was considered to be representative of a mixture of K and L subunits, some pyridoxalated, containing two POE molecules. Band 4 was always found to be a much tighter band than other bands on the gel and consistently migrated slightly above band 3. MALDI analysis indicated that it had a molecular weight corresponding to a subunit containing two POEs. Based upon the large breadth of the signal observed from MALDI analysis (data not shown), it was expected that the signal obtained was from POE modi¢ed hemoglobin. The band was known to contain hemoglobin since band 4 was detected on Western blots probed with anti-human hemoglobin antibody (for instance, see Fig. 8A). The MALDI-MS spectrum for band 5 possessed a single peak corresponding to mass 18 222. This molecular weight could correspond to an K subunit containing a POE but no pyridoxal-5-phosphate. Alter-
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5 was in£uenced by the amount of PEG added to the reaction. Reactions which contained a 20-fold excess of PEG had signi¢cantly more material which migrated slowly on the gels and only a small amount of material which migrated equivalently to unmodi¢ed hemoglobin. In contrast, addition of equimolar amounts of hemoglobin and PEG resulted in an elution pro¢le where the majority of the material eluted equivalently to unmodi¢ed hemoglobin and relatively little material migrated equivalently to modi¢ed hemoglobin in PHP. In general, the SDS-PAGE pro¢le of the 20UPEG-modi¢ed PLP-Hb was similar (rela-
Fig. 4. SDS-PAGE analysis of PEG hemoglobin. SDS-PAGE analysis of PHP compared to pyridoxalated hemoglobins containing various degrees of modi¢cation with monomethoxyPEG. Lane 1 contains PHP. The ratio of PEG to hemoglobin in the modi¢cations were 20U (lane 2), 10U (lane 3), 5U (lane 4), 1U (lane 5). Lane 6 contains pyridoxalated hemoglobin. Hemoglobin crosslinked between L subunits with a 3400 MW PEG is shown in lane 7. Lane 8 contains stroma-free hemoglobin. Lane 9 contains molecular weight markers.
natively, if the signal were due to a doubly charged species, the actual molecular weight would correspond to two subunits crosslinked with a POE molecule. No signal was detected by MALDI-MS for any of the protein species above band 5. In order to further assess the properties of modi¢ed hemoglobin subunits derivatized with POE, additional modi¢ed hemoglobin species were prepared. In this case, pyridoxalated stroma-free hemoglobin (PLP-Hb) was modi¢ed with 3000 MW PEG which was mono-functional (one end contained N-hydroxysuccinimide and the other a monomethoxy group). A series of modi¢ed hemoglobins were made in which the PEG to hemoglobin ratio in the reactions was 1:1, 5:1, 10:1 or 20:1. SDS-PAGE analysis of these derivatives is shown in Fig. 4 where lane 1 contained PHP and lanes 2^5 contained hemoglobins modi¢ed at the PEG:hemoglobin ratios listed above (1U to 20U). Lane 6 contained PLP-Hb and lane 8 contained stroma-free hemoglobin for reference. The pro¢le of the modi¢ed hemoglobin shown in lanes 2^
Fig. 5. SEC analysis of PEG hemoglobin. Pyridoxalated hemoglobin was reacted with molar excess (1U, 5U, 10U, or 20U) of monofunctional PEG and analyzed by SEC. PHP displays the bimodal distribution described previously. 1UPEG hemoglobin elutes slowly, indicating only minor modi¢cation. 5UPEG hemoglobin approximately co-elutes with the major peak of PHP. 10U and 20UPEG hemoglobin elute slightly more rapidly than the major peak of PHP, but not as rapidly as the high MW component of PHP. The presence of a small percentage of high molecular weight material in the 20U reaction is believed to be due to contamination of the monomethoxy-PEG with some bi-functional reagent.
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tive migration distance of the bands and the number of bands) to the pro¢le observed for PHP up to band 5, with the exception of band 4 in PHP. No tight band was observed in this PEG hemoglobin preparation. However in PEG hemoglobin preparations made at 1U and 5UPEG:hemoglobin ratios, a tight band which co-eluted with carbonic anhydrase in stroma-free hemoglobin (Lane 8) was observed. Small di¡erences in the migration distance of the PEG modi¢ed species were attributed to di¡erences in the size and functionality between POE and PEG. A hemoglobin species which contained a speci¢c crosslink between the L-93 cysteine residues was used as a standard to con¢rm the elution position of a crosslinked L subunit. Lane 7 on the gel contained this species (XL-Hb). The unmodi¢ed K subunits migrated at the gel front with a mobility equivalent to the 14 400 MW standard. A species which was not crosslinked, but which was derivatized with a monofunctional form of the PEG at the L-93 cysteine migrated equivalently to a species in the PHP with subunits containing one POE chain (band 2). The crosslinked L subunits migrated equivalently to band 5 in PHP. However, a band containing multiple PEGs in the 20U and 10UPEG reactions also migrated in the same region of the gel. It is not possible to distinguish the multi-PEG modi¢ed subunits from the crosslinked species on these SDS-PAGE gels. PHP contained signi¢cantly more high MW species, based upon migration distance, than the PEG modi¢ed hemoglobin. These species were postulated to be crosslinked subunits which also contained additional POE molecules attached at only one position. In theory, the distinct PHP bands observed in SDS-PAGE should correspond to globin subunits derivatized with 1, 2, 3, 4, or 5 POEs (MW V19 000, 24 000, 27 000, 30 000, and 33 000, respectively) and globin dimers (MW V35 000). However, MALDI-TOF analysis of isolated bands as well as analysis of hemoglobin derivatized with monofunctional PEG demonstrated that PEG-modi¢ed proteins migrate aberrantly in SDS-PAGE. Other observations of anomalies in migration of PEG-proteins during SDS-PAGE have been reported. For example, IL-2 molecules with 2 and 3 PEGs co-migrated on gels [40]. Size exclusion analysis of PHP and the PEG hemoglobin illustrated the di¡erence between highly
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modi¢ed species and crosslinked species. Fig. 5 shows an overlay of chromatograms for PHP and the PEG-modi¢ed material. Even in derivatives which were synthesized with 10U or 20U excess of PEG to hemoglobin, the elution pro¢les were monomodal. The randomly PEG-modi¢ed hemoglobins were eluted from the SEC column after the population of high molecular weight species in PHP. Hemoglobin reacted with a 5U excess of PEG eluted in the same region of the chromatogram as the lower molecular weight peak of PHP. Based upon the elution pattern of the PEG hemoglobin, the lower MW peak of PHP was interpreted as hemoglobin tetramers with varying degrees of derivatization with POE. The higher MW portion of PHP was deduced to be tetramers joined to other tetramers with a POE crosslink. The SEC chromatogram shown in Fig. 5 illustrates a key di¡erence in the structure of proteins modi¢ed with a mono- vs bi-functional PEG molecule. Higher order tetramers are found in preparations made using a bi-functional reagent. If the chemical modi¢cation conditions are not tightly regulated, large networks of polymerized protein molecules can occur (leading to gelation of the proteins in some cases (data not shown)).
Fig. 6. IEF analysis of PHP. PHP focused as a broad band with an average pI of 5.8 on 3^9 pH gels. Unmodi¢ed hemoglobin focused as a tight band with a pI of approximately 7.1.
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Fig. 7. Western blots of PHP. Each of the panels shown contains a gel with a lane with (1) puri¢ed enzyme, (2) PHP and (3) molecular weight markers. (A) A Western blot probed with: anti-human catalase, (B) anti-human SOD, and (C) anti-human carbonic anhydrase. In each panel, the enzyme content of the PHP has been modi¢ed to some extent by derivatization with POE.
In addition to SDS-PAGE, PHP species were also examined by IEF analysis. PHP appeared as a broad band between protein standards of known isoelectric points (5.0^6.5) as shown in Fig. 6. The average isoelectric point of PHP was estimated to be 5.8. The pI of unmodi¢ed hemoglobin, which co-migrates with the hemoglobin contained in the standard protein mix, is approximately 7.1. No attempt to quantify any of the species on the Coomassie blue stained gels (SDS-PAGE or IEF) was made due to previous reports that PEG modi¢ed proteins were not stained quantitatively compared to their non-PEG-modi¢ed counterparts [40]. The hydrodynamic radii (RH ) of PHP and other hemoglobin solutions were determined by dynamic light scattering on a DynaPro Molecular Sizing Instrument. The puri¢ed hemoglobin generated a narrow monomodal distribution with a mean RH of 3.2 nm and a standard deviation of 0.5 nm. The average RH for ¢ve lots of PHP was 7.2 þ 0.6 nm. All ¢ve PHP samples had broad unimodal distributions with average standard deviation values of 2.1 nm. These molecular sizes for PHP and hemoglobin agree with
those reported by Vandegri¡ et al. (RG = 7.2 and 2.7 nm, respectively) [42] determined by measuring the colloidal osmotic pressure of the proteins in solution. PHP is a heterogeneous mixture of di¡erent forms of derivatized hemoglobin subunits which can be manufactured reproducibly and has been shown to be safe through pre-clinical and clinical trials. Based upon the manufacturing process, PHP was expected to contain many of the soluble enzymes which are found together with hemoglobin in red blood cells. The presence of some of these enzymes in PHP has been demonstrated by direct assay of their activities in PHP [43]. In addition, an immunoblot of PHP con¢rmed the presence of the enzymes catalase, SOD and carbonic anhydrase (Fig. 7). The enzymes were also derivatized with polyoxyethylene as evidenced by their increased molecular weight as compared to unmodi¢ed protein standards on the blots. The presence of enzymes, such as catalase, in PHP preparations may be a signi¢cant factor in controlling the oxidation rate of the heme groups [11,43] or may have bene¢cial e¡ects in treatment during certain disease states. Catalase and superoxide dismu-
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Fig. 8. Western blot of fractions from a¤nity chromatography. (A) An immunoblot probed with anti-human hemoglobin antibody. The load and unbound fractions have similar pro¢les, but the bound material is enriched for high molecular weight hemoglobin components. (B) An immunoblot probed with anti-human catalase antibody. Load, unbound and bound fractions possess similar pro¢les.
Fig. 9. PHP in solution. PHP is composed of tetrameric, pyridoxalated subunits bound to POE. A fraction of the tetramers are linked to other tetramers (and other red cell proteins) by POE, which is a bifunctional reagent capable of reacting with primary amines. The tetramers (of crosslinked and non-crosslinked species) are free to dissociate and re-associate depending on the solution characteristics.
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tase combinations have been evaluated for scavenging reactive oxygen species in animals [44]. The exogenous addition of superoxide dismutase and catalase to hemoglobin-based pharmaceuticals has been suggested for treatment of ischemia reperfusion injury and as a method to decrease hemoprotein-associated free radical events following oxidant challenge [45^ 48]. The enzymes were not only expected to be derivatized with POE, but were also expected to be attached to other proteins due to the bifunctional nature of POE. This was con¢rmed for catalase by examining the catalase-containing species in PHP. An a¤nity column was made with anti-catalase antibody, and PHP was applied to the column. The column was washed extensively with bu¡er to remove any non-speci¢cally bound material and the catalase containing material which was bound to the column was eluted with pH 2.5 glycine bu¡er. Samples of the load, non-bound (the £ow through) and bound fractions were separated in duplicate on a SDS-PAGE gel and blotted to nitrocellulose. One set of samples was probed with anti-hemoglobin antibody, while the other set was probed with anti-catalase antibody. The results are shown in Fig. 8. The blots probed with anti-catalase antibody (Fig. 8B) indicated that much catalase-containing material £owed through the column (non-bound fraction). However, catalase containing material was also speci¢cally bound and eluted with the low pH wash. The distribution of catalase species in load, unbound and bound fractions were all similar. The blots probed with antihemoglobin antibody (Fig. 8A), showed that the pro¢les of load and unbound fractions were similar, but that the bound material was enriched for higher molecular weight hemoglobin species. This high molecular weight hemoglobin represented material which was crosslinked to catalase by POE. The tethering of these potentially bene¢cial enzymes to hemoglobin would not occur if the conjugate were mono-functional PEG. Only a bifunctional molecule, such as the POE used here, will result in crosslinks between proteins. The crosslink ensures that these tethered enzymes are co-distributed with PHP within the body. However, at this time, the tissue distribution of the di¡erent species in PHP has not been elucidated. PHP is a complex mixture of di¡erent derivatives
of hemoglobin. Based upon the chemical characterization of PHP and the properties of the chemical modi¢cation agents, our current interpretation of the structure of PHP is shown in Fig. 9. The product primarily consists of modi¢ed tetrameric hemoglobin with a portion existing as crosslinked tetrameric species. Less than 1% of the tetramers are expected to be found as unmodi¢ed species. The tetrameric species can dissociate into dimers while the dimers can reassociate with other modi¢ed or unmodi¢ed dimers to reform tetramers. Many of the other soluble proteins found in the red blood cell are also found in PHP. These proteins are also modi¢ed by the attachment of POE and are likely to be crosslinked to PHP. The safety of PHP has been extensively studied in a number of animal models and is currently being evaluated in human clinical trials [28,49,50]. Acknowledgements We thank Dr. Dan Snyder of Protein Solutions for the dynamic light scattering measurements. Thanks also to Dr. Carol Haney and N. Srinivasan of The North Carolina State University for MALDI-MS analyses. We also thank Dr. Teresa Keng and Dr. Chris Privalle for critical review of the manuscript and Dr. Yuji Iwashita for helpful discussions about PHP.
References [1] R.E. Dickerson, I. Geis, Hemoglobin: Structure, Function, Evolution, and Pathology, Benjamin/Cummings, Menlo Park, 1983. [2] M.F. Perutz, G. Fermi, Haemoglobin and Myoglobin, Atlas of Molecular Structures in Biology, Vol. 2, Oxford University Press, New York, 1981. [3] T. Suzuki, T. Takagi, S. Ohta, Biochem. J. 260 (1989) 177^ 182. [4] A. Fago, E. Bendixen, H. Malte, R.E. Weber, J. Biol. Chem. 272 (1997) 15628^15635. [5] R.C. Hardison, D.H.K. Chui, C.R. Riemer, W. Miller, M.F.H. Carver, T.P. Molchanova, G.D. Efremov, T.H.J. Huisman, Hemoglobin 22 (1998) 113^127. [6] V.S. Sharma, T.G. Traylor, R. Gardiner, Biochemistry 26 (1987) 3837^3843. [7] M. Brunori, R.W. Noble, E. Antonini, J. Wyman, J. Biol. Chem. 225 (1966) 5238^5243.
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T.L. Talarico et al. / Biochimica et Biophysica Acta 1476 (2000) 53^65 [8] L. Stryer, Biochemistry, W.H. Freeman, San Francisco, 1981. [9] B. Halliwell, J.M. Gutteridge, Methods Enzymol. 186 (1990) 1^85. [10] A.I. Alayash, B.A. Brockner Ryan, R.E. Cashon, Arch. Biochem. Biophys. 349 (1998) 65^73. [11] T. Talarico, A. Swank, C. Privalle, Biochem. Biophys. Res. Commun. 250 (1998) 354^358. [12] T. Yang, K.W. Olsen, Artif. Cells Blood Substitutes Immobilization Biotechnol. 22 (1994) 709^717. [13] M.I. Covas, L. Coca, C. Ricos, J. Marrugat, Clin. Chem. 43 (1997) 1991^1993. [14] A. Mansouri, C.A. Perry, Hemoglobin 11 (1987) 353^371. [15] G. Reah, A.R. Bodenham, A. Mallick, E.K. Daily, R.J. Przybelski, Crit. Care Med. 25 (1997) 1480^1488. [16] S.A. Gould, E.E. Moore, D.B. Hoyt, J.M. Burch, J.B. Haenel, J. Garcia, R. DeWoskin, G.S. Moss, J. Am. Coll. Surg. 187 (1998) 113^122. [17] R.J. Przybelski, E.K. Daily, J.C. Kisicki, C. Mattia Goldberg, M.J. Bounds, W.A. Colburn, Crit. Care Med. 24 (1996) 1993^2000. [18] S. Kasper, M. Walter, F. Grune, A. Bischo¡, H. Erasmi, W. Buzello, Anesth. Analg. 83 (1996) 921^927. [19] J.R. Hess, Curr. Opin. Hematol. 3 (1996) 492^497. [20] M.G. Scott, D.F. Kucik, L.T. Goodnough, T.G. Monk, Clin. Chem. 43 (1997) 1724^1731. [21] J.P. Savitsky, J. Doczi, J. Black, J.D. Arnold, Clin. Pharmacol. Ther. 23 (1978) 73^80. [22] J.R. Hess, Sem. Hematol. 33 (1996) 369^378. [23] Y. Iwashita, A. Yabuki, K. Yamaji, K. Iwasaki, T. Okami, C. Hirata, K. Kosaka, Biomat. Artif. Cells Artif. Organs 16 (1988) 271^280. [24] R. Kluger, J. Wodzinska, R.T. Jones, C. Head, T.S. Fujita, D.T. Shih, Biochemistry 31 (1992) 7551^7559. [25] R. Chatterjee, E.V. Welty, R.Y. Walder, S.L. Pruitt, P.H. Rogers, A. Arnone, J.A. Walder, J. Biol. Chem. 261 (1986) 9929^9937. [26] Y.J. Iwashita, in: E. Tsuchida (Ed.), Arti¢cial Red Cells, John Wiley and Sons, 1995, pp. 151^176. [27] R.G. Kilbourn, J. DeAngelo, in: Schlag, Redl (Eds.), Shock, Sepsis and Organ Failure, Springer-Verlag, Heidelberg, 1999. [28] H.G. Bone, P.J. Schenarts, S.R. Fischer, R. McGuire, L.D. Traber, D.L. Traber, J. Appl. Physiol. 84 (1998) 1991^ 1998. [29] R. Benesch, R.E. Benesch, Methods Enzymol. 76 (1981) 147^158. [30] M.L. Nucci, R. Schorr, A. Abuchowski, Adv. Drug Deliv. Rev. 6 (1991) 133^151.
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[31] F.M. Veronese, P. Caliceti, A. Pastorino, O. Schiavon, L. Sartore, L. Banci, L.M. Scolaro, J. Control. Release 10 (1989) 145^154. [32] S.P. Monkarsh, Y. Ma, A. Aglione, P. Bailon, D. Ciolek, B. DeBarbieri, M.C. Graves, K. Hollfelder, H. Michel, A. Palleroni, J.E. Porter, E. Russoman, S. Roy, Y.C. Pan, Anal. Biochem. 247 (1997) 434^440. [33] R. Clark, K. Olson, G. Fuh, M. Marian, D. Mortensen, G. Teshima, S. Chang, H. Chu, V. Mukku, E. Davis, T. Somers, M. Cronin, M. Winkler, J.A. Wells, J. Biol. Chem. 271 (1996) 21969^21977. [34] H. Sato, M. Watanabe, Y. Iwashita, Bioconjugate Chem. 6 (1995) 249^254. [35] S. Matsumura, K. Yamaji, H. Ohki, K. Kosaka, Y. Iwashita, Biomat. Artif. Cells Immobilization Biotechnol. 20 (1992) 435^438. [36] T.L. Talarico, K. Guise, V.K. Garg, Biopharmacology 12 (1999) 42^47. [37] B. Skoog, Vox Sang 37 (1979) 345^349. [38] X. Liang, J. Bai, Y. Liu, D.M. Lubman, Anal. Chem. 68 (1996) 1012^1018. [39] P.M. Miles, K.V. Langley, C.J. Stacey, T.L. Talarico, Artif. Cells Blood Substitutes Immobilizaton Biotechnol. 25 (1997) 315^326. [40] M. Kunitani, G. Dollinger, D. Johnson, L. Kresin, J. Chromatogr. 588 (1991) 125^137. [41] P. McGo¡, A.C. Baziotis, R. Maskiewicz, Chem. Pharm. Bull. 36 (1988) 3079^3091. [42] K.D. Vandegri¡, M. McCarthy, R.J. Rohlfs, R.M. Winslow, Biophys. Chem. 69 (1997) 23^30. [43] C. Privalle, T. Keng, J. DeAngelo, T. Talarico, Free Radic. Biol. Med. 25 (Suppl. 1) (1998) S65. [44] Y. Yabe, M. Nishikawa, A. Tamada, Y. Takakura, M. Hashida, J. Pharmacol. Exp. Ther. 289 (1999) 1176^1184. [45] F. D'Agnillo, T.M.S. Chang, Biomat. Artif. Cells Immobilization Biotechnol. 21 (1993) 609^621. [46] F. D'Agnillo, T.M.S. Chang, Free Radic. Biol. Med. 24 (1997) 906^912. [47] S. Razack, F. D'Agnillo, T.M.S. Chang, Arti¢c. Cells Blood Substitutes Immobilization Biotechnol. 25 (1997) 181^192. [48] F. D'Agnillo, T.M.S. Chang, Artif. Cells Blood Substitutes Immobization Biotechnol. 25 (1997) 163^180. [49] M. Matsushita, A. Yabuki, J.F. Chen, T. Takahashi, H. Harasaki, P.S. Malchesky, Y. Iwashita, Y. Nose, H. Asaio, Transactions 34 (1988) 280^283. [50] T. Takahashi, K. Iwasaki, P.S. Malchesky, H. Harasaki, M. Matsushita, Y. Nose, H. Rolin, P.M. Hall, Artif. Organs 17 (1993) 153^163.
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Chemical characterization of pyridoxalated hemoglobin polyoxyethylene conjugate Todd L. Talarico *, Katherine J. Guise, Cyrus J. Stacey Apex Bioscience Inc., P.O. Box 12847, Research Triangle Park, NC 27709, USA Received 2 July 1999; received in revised form 14 September 1999; accepted 16 September 1999
Abstract Pyridoxalated hemoglobin polyoxyethylene conjugate (PHP) was developed in the 1980s as an oxygen carrier and is now under development for treatment of nitric oxide-dependent, volume refractory shock. PHP is made by derivatizing human stroma-free hemoglobin with pyridoxal-5-phosphate and polyoxyethylene (POE). A unique aspect of using POE for modification is that unlike its mono-methoxy polyethylene glycol (PEG) relatives, POE is bifunctional. The result of derivatization of stroma-free hemoglobin is a complex mixture of modified hemoglobin and other red cell proteins. The molecular weight profile, based on size exclusion chromatography, is bimodal and has a number average molecular weight of approximately 105 000 and a weight average molecular weight of approximately 187 000. The mixture of hemoglobin molecules has on average 3.3 pyridoxal and 5.0 polyoxyethylene units per tetramer. A portion of the tetramers are linked by POE crosslinks. The hemoglobin tetramers retain their ability to dissociate into dimer pairs and only a small percentage of the dimer pairs are not modified with POE. The SDS-PAGE profile exhibits the ladder-like appearance commonly associated with polyethylene glycol-modified proteins. The isoelectric focusing profile is broad, demonstrating a pI range of 5.0^6.5. The hydrodynamic size of PHP was determined to be approximately 7.2 nm by dynamic light scattering. Soluble red blood cell proteins, such as catalase, superoxide dismutase, and carbonic anhydrase, are present in PHP and are also modified by POE. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Hemoglobin; Polyethylene glycol; Polyoxyethylene; Blood substitute; Shock
1. Introduction Hemoglobin accounts for greater than 90% of soluble protein in a red blood cell and is responsible for the transport of oxygen from the lungs to other tissues in the body [1]. Hemoglobin Ao and its variants from humans and hemoglobins from a large number of organisms have been studied extensively [2^5]. The crystal structures of a number of di¡erent
* Corresponding author. Fax: +1-919-405-4010; E-mail: [email protected]
hemoglobins have been determined and their function in transport of oxygen, carbon dioxide and nitric oxide has been elucidated [2,6,7]. Inside red blood cells, human hemoglobin Ao exists as a tetramer containing 2 K and 2 L subunits. Stable dimers consisting of an K and L subunit are in equilibrium with the tetramer form in solution and the dissociation state of the tetramers depends on solution characteristics, such as hemoglobin concentration, salt content and pH. Each of the subunits contains an iron protoporphyrin which must exist in the ferrous state in order for hemoglobin to transport oxygen [8]. During the course of routine functioning in the
0167-4838 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 9 ) 0 0 2 1 1 - 3
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body, hemoglobin is exposed to a number of insults which result in the oxidation of the ferrous form of the iron to the ferric form. These insults include exposure to hydrogen peroxide, peroxynitrite and nitric oxide [9^11]. These insults are opposed by antioxidant enzymes and reductases [12,13], which e¤ciently maintain more than 99% of the red cell hemoglobin in the ferrous state [14]. A number of hemoglobin-based therapeutics are currently under development by companies in the United States and abroad [15^18]. Hemoglobin therapeutics have been designed as oxygen-carrying £uids useful for blood replacement during surgery and in trauma, enhancers of radiation therapy, and scavengers of nitric oxide [19,20]. Hemoglobin, once removed from the red cell, cannot be used for these therapeutic indications without modi¢cation due to renal toxicity, as described by Savitsky et al. [21]. In addition, the plasma half-life of unmodi¢ed hemoglobin is short and the oxygen a¤nity of hemoglobin in plasma may be too great to allow e¤cient delivery of oxygen in those applications where oxygen delivery is desired [22]. These limitations to the use of unmodi¢ed human hemoglobin have been circumvented by chemical modi¢cation of the hemoglobin in one or more of the following ways: (1) crosslinking K, L dimers; (2) polymerizing hemoglobin; or (3) binding polymers to the surface of hemoglobin [23^ 25]. One such product, pyridoxalated hemoglobin polyoxyethylene conjugate (PHP) was initially developed in the 1980s as an oxygen-carrying solution [26]. Currently, PHP is in clinical trials for treatment of nitric oxide-dependent, volume refractory shock [27,28]. PHP is produced from erythrocyte lysate made from outdated human red blood cells. The method used provides for separation of the hemoglobin from red cell stroma and potential adventitious agents, but not from many of the enzymes naturally associated with hemoglobin in red cells. The hemoglobin is pyridoxalated to reduce the oxygen a¤nity [29]. The pyridoxalated stroma-free hemoglobin is then modi¢ed with the homo-bifunctional molecule POE to increase the hydrodynamic volume (apparent molecular weight) of the molecule. The resulting modi¢ed hemoglobin entity is puri¢ed to remove residual reactants, formulated in electrolytes, and rendered sterile for use as a parenteral drug.
This conjugated hemoglobin has unique characteristics conferred by the decoration with POE. Currently, there are a number of proteins conjugated with PEG which have been approved by the Food and Drug Administration for human use [30]. The chemical characterization of many PEG-conjugated proteins has been described [30^33], but there are few examples of characterization of POE-conjugated proteins. Among these, Sato et al. reported on the characterization of superoxide dismutase-POE-deferoxamine and Iwashita performed some preliminary characterization of POE-modi¢ed hemoglobin (PHP)[26,34,35]. Since PHP will be used as a large volume parenteral drug in a population which is typically critically ill, understanding the safety and biochemical nature of the product is important. This report describes the chemical characterization of PHP. The characterization provides new information about the nature of the chemical modi¢cation and the association of the subunits of the molecule as they exist in solution. The association of subunits with other proteins also occurs due to the unique properties of the modi¢cation agent used during production of PHP. 2. Materials and methods PHP was produced by the Apex Bioscience Manufacturing group based upon the synthesis method described by Iwashita and colleagues [35]. The production process was modi¢ed as described by Talarico et al. [36]. All PHP used in this study met the requirements for release by the Apex Quality Control group and were representative of material used in clinical trials. The K,K-crosslinked hemoglobin was a gift from the Walter Reed Army Institute of Research. In addition to PHP, several hemoglobin derivatives were synthesized and analyzed. Pyridoxalated hemoglobin was synthesized by reaction of excess pyridoxal-5-phosphate [29] with deoxygenated stroma-free hemoglobin solution and was reduced with sodium borohydride (Alfa Aesar, Ward Hill, MA). PEG hemoglobin was synthesized using monomethoxy polyethylene glycol (MW 3000) activated with N-hydroxysuccinimide (Shearwater Polymers, Huntsville, AL). Various molar excesses of PEG to pyri-
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doxalated hemoglobin were reacted in bu¡er containing 50 mM sodium phosphate and sodium chloride, pH 8.0 for 1 h at room temperature. The reaction mix was dialyzed at 4³C for 24 h against phosphate bu¡er to remove residual reactants. Crosslinks speci¢c to L-93 cysteine were made on pyridoxalated hemoglobin with a bifunctional maleimide PEG, MW 3400 (Shearwater Polymers). The reaction was performed with a 1.5-fold molar excess of crosslinker to hemoglobin at room temperature for 3 h. The reaction mix was dialyzed against phosphate bu¡er to remove residual reactants. Size exclusion chromatography (SEC) for molecular weight determination was performed on a Waters Alliance 2690 separations module with detection at A280 (Waters, Milford, MA). All data were analyzed with Waters Millenium software equipped with the GPC processing package. The separation was performed at 30³C on a BioSEP-S-4000 column (Phenomenex, Torrance, CA) using a 0.1 M potassium phosphate mobile phase, pH 6.8, containing 13% ethanol. Ten microliters of a 30 mg/ml solution of PHP was injected and the molecular weight of the sample was determined from a standard curve derived from the retention times of protein reference standards (Bio-Rad, Hercules, CA). SEC for estimation of the percentage of unmodi¢ed dimer was performed on a 410 series bio LC pump (Perkin-Elmer, Norwich, CT) equipped with a Rheodyne injector and a Waters 990 photodiode array detector at A280 . All data were analyzed with the software package supplied with the detector. The separation was accomplished with 20 Wl of a 5 mg/ml sample on a SuperDex 75 column (Pharmacia Biotech, Piscataway, NJ) using a 25 mM Tris HCl pH 8.0/1.0 M magnesium chloride mobile phase at a £ow rate of 0.5 ml/min. Hemoglobin and methemoglobin concentrations were determined using an IL 482 CO-Oximeter (Instrumentation Laboratory, Lexington, MA). The extent of pyridoxalation was determined following digestion of PHP to liberate the phosphate. PHP was dialyzed against USP puri¢ed water and 25 mg of PHP was placed in a glass test tube. Digest solution composed of sodium molybdate, sulfuric acid and perchloric acid was added to the tube and the mix was heated at 180³C for 60 min. The phosphate content was then determined using a kit for
55
determination of inorganic phosphate according to the manufacturers instructions (Sigma, St. Louis, MO, Procedure 670). The determination of the extent of modi¢cation of the hemoglobin with POE was performed after digestion of the PHP with 6 N HCl for 6 h at 110³C to liberate the POE. The POE content of the digest solution was determined by the method of Skoog [37] by comparison to POE standards (Nippon Oil and Fats, Tokyo, Japan). Titrisol reagent was obtained from Merck (Germany). Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of PHP and other hemoglobin species was performed on 10% NuPAGE gels (Novex, San Diego, CA) under reducing conditions. The separation was developed with 3-(N-morpholino)ethane sulfonic acid-SDS running bu¡er as described by the manufacturer on the gel insert instructions. Molecular weight standards (Mark 12, Novex) were included on each gel. Proteins were visualized by staining with colloidal blue Coomassie G-250 (Novex) according to the manufacturer's instructions. Quantitation of protein bands was performed by scanning the gel using a pdi 420oe scanner (BioRad) and analyzing each lane with Quantity One software. Western blots were performed using antibodies to the following human enzymes: Cu/Zn superoxide dismutase (SOD), catalase, and carbonic anhydrase II (The Binding Site, Birmingham, UK). SDS-PAGE separation was achieved on 10% NuPAGE gels as described above. Depending on the sensitivity of the antibody, PHP was loaded onto the gel at 10^ 50 Wg hemoglobin. Human erythrocyte enzymes corresponding to each antibody were obtained from Sigma (St. Louis, MO) to serve as positive controls. The load of each enzyme was based upon the antibody sensitivity, and ranged from 10 to 50 ng. A prestained molecular weight marker (SeeBlue, Novex) was also included with each blot to assess transfer e¤ciency. The proteins were blotted onto nitrocellulose membranes (Novex) in Tris-Glycine transfer bu¡er. Upon completion of the transfer, the membranes were blocked in 3% milk in 1UTris bu¡ered saline (TBS) for 1.5^2 h. The primary antibodies were diluted 1:1000 in 1% milk in 1UTBS (50 Wl in 50 ml) with or without 0.025% Tween-20 (BioRad) and incubated overnight at 42³C. The mem-
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branes were washed in 1UTBS with 0.025% Tween20 then placed in secondary antibody. The membranes were incubated in peroxidase conjugated donkey anti-sheep/goat IgG (HpL) (The Binding Site), diluted in the same manner as the primary antibody, for approximately 3 h at 42³C, followed by another set of TBS/Tween-20 washes. Visualization of catalase and carbonic anhydrase was accomplished with tetramethyl benzidine (TMB) substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD). The SOD blot was developed in SuperSignal Chemiluminescent substrate (Pierce, Rockford, IL). The blot was exposed to Kodak Biomax ¢lm (Rochester, NY) for 10 s and processed using a Konica (Japan) QX-70 processor. IEF analysis was performed on 3^9 IEF gels according to the manufacturer's directions (Novex). IEF 3^9 standards were used for calibration (BioRad). Gels were stained with Coomassie blue and quantitated as described for the SDS-PAGE analysis. Hydrodynamic size measurements were performed on a DynaPro Molecular Sizing Instrument (Protein Solutions, Charlottesville, VA). PHP samples were diluted to approximately 11 mg/ml in 100 mM sodium phosphate, pH 7.2. Hemoglobin samples were diluted to approximately 6 mg/ml in the same bu¡er. All samples were ¢ltered prior to injection with a 100-nm Whatman Anodisc Membrane (Whatman, Clifton, NJ) for particulate removal. Samples were analyzed at 20³C, and scattering signals were collected at 90³ from the incident laser beam. Hydrodynamic size distributions were generated from the resulting autocorrelation function with DynaLS Non-negative least squares algorithm (Copyright 1996, 97 Alexander Goldin, Nickolay Sidorenko). The result provided the mean peak size and standard deviation for each peak resolved. Catalase and derivatized catalase in PHP were puri¢ed by a¤nity chromatography. A¤nity resin was prepared by attachment of anti-human erythrocyte catalase antibody (The Binding Site) to CNBr Sepharose CL-4B (Pharmacia Biotech) as recommended by the manufacturer. A 200-mg PHP sample in 0.1 M Tris-HCl, pH 8.0/0.5 M NaCl was passed through a 1-ml column of the resin. Bound material was eluted in 200 mM glycine HCl and immediately neutralized with 1 M Tris HCl, pH 8.0. The elution was performed with a peristaltic pump (Master£ex, Cole-
Parmer, Chicago, IL), monitored at A280 with a UV detector (Pharmacia Biotech) and recorded on a strip chart recorder. The collected fractions were separated by SDS-PAGE, blotted onto nitrocellulose, and protein species were detected with antibody to hemoglobin (Chemicon, Temecula, CA) or catalase as described above. MALDI mass spectrometry (MS) analysis was performed on PHP which had been fractionated by SDS-PAGE and then blotted onto nitrocellulose. For analysis, PHP was separated on a 10% Tris-glycine gel (Novex), blotted onto nitrocellulose, and stained with Ponceau S to visualize the protein bands. Slices of the membrane corresponding to distinct protein bands were cut from the blot using a razor blade and were dissolved in acetone for application to the MALDI target [38]. The MALDI investigations were performed using a Bruker Pro£ex+ (Bruker Daltronics, Billerica, MA) linear MALDI-TOF instrument with a 1.2-m £ight tube equipped with a nitrogen laser. Detection was performed with a dual microchannel plate detector at a typical detector voltage of 1.55 V. A sampling rate of 1 GHz and pulsed ion extraction were used during data collection. The data were collected and processed with the Xacq 3.0 and Xmass 3.0 software packages (Bruker). 3. Results and discussion The molecular weight (MW) of PHP was estimated by SEC based upon retention time as compared to protein standards. Nine manufacturing lots of PHP were analyzed and the peak average MW was determined to be 106 164 þ 3385, the number average MW was determined to be 105 122 þ 3566 and the weight average MW was determined to be 187 000 þ 8300. The pro¢le of the chromatogram indicated that the MW distribution was bimodal as shown in Fig. 1. The polydispersity of the PHP was 1.778 þ 0.031. Typical peak molecular weights from the two main species of the bimodal distribution were 106 000 and 300 000. The relative peak areas were typically on the order of 70% (low MW component) and 30% (high MW component) when approximated using a drop baseline for integration. Stroma-free hemoglobin had a peak MW of 38 214 and hemoglobin crosslinked
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Fig. 1. SEC chromatogram of PHP and unmodi¢ed hemoglobin. PHP displays a bimodal distribution. Unmodi¢ed hemoglobin elutes after the majority of PHP.
between the K subunits had a peak MW of 39 288 when analyzed on the same chromatography system. Derivatization of hemoglobin to produce PHP includes attachment of POE and pyridoxal-5-phosphate. POE was released from PHP by digestion with hydrochloric acid and quantitated spectrophotometrically after it was complexed with barium/iodine. The analysis of ¢ve lots of PHP demonstrated that on average 5.0 þ 0.4 POE molecules were attached to each hemoglobin tetramer. This number agreed with the estimated extent of PHP derivatization based upon chromatographic analyses performed for process POE mass balances [39]. However, Iwashita et al. reported the presence of approximately 6 mol of POE per mol of hemoglobin in a previous study [26]. Future studies to examine the POE content of PHP using a chromatographic quantitation technique may resolve the reported differences [39]. The number of pyridoxal-5-phosphate molecules attached was determined by digestion of PHP followed by assay for inorganic phosphate con-
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tent. Each hemoglobin tetramer was found to contain 3.3 þ 0.1 pyridoxal-5-phosphate molecules based upon the analysis of ¢ve lots of PHP. This number was in agreement with a previous report by Iwashita et al. [26]. The relative percentages of POE-modi¢ed to unmodi¢ed K^L dimer pairs was determined by SEC performed under dissociating conditions where the SEC mobile phase contained 1 M magnesium chloride to promote dimer dissociation. The position of the unmodi¢ed dimer peak was con¢rmed by co-elution with puri¢ed hemoglobin and by performing studies in which puri¢ed hemoglobin was added to PHP samples. The percentage of unmodi¢ed dimer pairs in nine lots of PHP was determined to be 6.6 þ 1.3% with a range from 4.9 to 8.6%. The ability of PHP to separate into dimer pairs illustrates one di¡erence between this class of modi¢ed hemoglobins and those that are crosslinked between dimer pairs (such as K,K-crosslinked hemoglobin). PHP retains the ability to form an equilibrium mix of species in solution because its dimer pairs are free to dissociate. This equilibration between di¡erent species was demonstrated by addition of unmodi¢ed hemoglobin to PHP. Fig. 2 contains SEC chromatograms for PHP performed under nondissociating conditions (as was shown in Fig. 1). Fig. 2A represents SEC chromatograms on a series of samples in which puri¢ed hemoglobin was added to PHP to make a ¢nal concentration of 0, 5, 10 and 20% with respect to the puri¢ed hemoglobin. Additions of hemoglobin to PHP were performed such that the total hemoglobin concentration in samples and amount of hemoglobin loaded on the SEC column were identical in all analyses. The series of chromatograms show a progressive decrease in the two main peak heights and a progressive increase in the region of lower molecular weight hemoglobin. As a result of the additions, the overall retention time of all the PHP species shifted to longer and longer retention times. The PHP sample spiked with 20% hemoglobin possessed a distinct shoulder which co-migrated with puri¢ed hemoglobin (see Fig. 2C). Additions of hemoglobin to 5 or 10% did not result in formation of a distinct shoulder on the chromatograms but resulted in the broadening of the molecular weight distribution. The broadening can be noted by following the migration of the back-end
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In contrast to the chromatograms generated after addition of unmodi¢ed hemoglobin, the addition of K,K-crosslinked hemoglobin to PHP resulted in the chromatograms shown in Fig. 2B. In this case, dimer pairs of the K,K-crosslinked hemoglobin could not dissociate and therefore could not recombine with PHP. Additions of crosslinked hemoglobin into PHP solution to ¢nal concentrations of 0, 5, 10 and 20% resulted in a peak which co-eluted with K,K-crosslinked hemoglobin injected separately (Fig. 2C). The series of chromatograms show a progressive decrease in the two main peak heights and a progressive increase in the peak corresponding to K,K-crosslinked hemoglobin. The addition of crosslinked hemoglobin to PHP did not increase the peak retention time of the two main PHP peaks in the chromatogram. This indicated that the PHP and K,K-crosslinked hemoglobin did not undergo subunit exchange. Based upon the chromatograms generated by ad-
Fig. 2. SEC chromatograms of PHP spiked with hemoglobin. (A) Chromatograms representing PHP spiked with unmodi¢ed hemoglobin at ¢nal contents of 0, 5, 10 and 20% of the total hemoglobin concentration. The changes observed in the chromatograms proceed in order corresponding to the unmodi¢ed hemoglobin additions from 0 to 20% as shown. (B) Chromatograms representing PHP spiked with K,K-crosslinked hemoglobin at ¢nal contents of 0, 5, 10 and 20% of the total hemoglobin concentration. (C) Chromatograms representing unmodi¢ed hemoglobin and K,K-crosslinked hemoglobin analyzed in the absence of PHP. For the labeling on the chromatograms 0U is 0% and 20U is 20%.
of the chromatogram in Fig. 2A as higher levels of unmodi¢ed hemoglobin are added to PHP. This observation is consistent with dissociation and re-arrangement between dimers in PHP and puri¢ed hemoglobin; unmodi¢ed dimers exchanged with the PHP dimers would result in new species of PHP molecules with lower molecular weights.
Fig. 3. SDS-PAGE analysis of PHP on 10% gels. PHP exhibits the ladder pattern typical of PEG modi¢ed proteins. The fastest migrating band corresponds to the migration of K and L subunits which do not contain POE (band 1). Bands 2 and 3 correspond to subunits with 1 or 2 POE units. Band 4 is a tight band that has not been conclusively identi¢ed. Bands 5 and greater are apparent either as major individual species or as a smear at the most slowly migrating positions.
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dition of hemoglobin solutions to PHP, it should be possible to predict the limit of detection of unmodi¢ed hemoglobin in PHP solutions using the SEC technique. If it was assumed that unmodi¢ed dimers combined with modi¢ed and unmodi¢ed subunits with equal preference and there are approximately 6.6% unmodi¢ed dimers present in PHP solutions, one would expect 0.066U0.066 occurrences of unmodi¢ed tetramer. Such a scenario would lead to approximately 0.4% unmodi¢ed tetramer in a typical PHP lot. Based upon the spiking experiments performed with PHP, unmodi¢ed tetramers present at 0.4% would not be detectable in PHP solutions. Even additions of unmodi¢ed hemoglobin to 20% of the total hemoglobin content in PHP solutions would only be expected to yield approximately 4% unmodi¢ed tetramer when dimers were free to recombine with modi¢ed hemoglobin. Interestingly, a 20% spike of unmodi¢ed hemoglobin can be detected as a distinct shoulder on the PHP peak, similar to a 5% spike of K,K-crosslinked hemoglobin. The modi¢cation of hemoglobin at the K and L subunit level was visualized by performing SDSPAGE analysis and staining with Coomassie blue. A typical gel is shown in Fig. 3 and the bands corresponding to di¡erent protein species are numbered. The pro¢le of the material appeared ladder-like where K and L subunits which were not modi¢ed with POE migrated with approximately the same mobility as the protein hen egg white lysozyme (MW = 14 400). The heterogeneity of the low molecular weight species was due to the pyridoxalation of the hemoglobin subunits (see Fig. 4, lanes 6 and 8). Similar ladder-like pattern of migration of the higher MW bands of PHP has been observed for a number of PEG derivatized proteins [40,41]. Table 1 MALDI-TOF analysis of hemoglobin and PHP separated by SDS-PAGE Sample
Band number
Molecular weight
Hemoglobin
1
PHP
1
PHP PHP PHP PHP
2 3 4 5
15324 16090 15422 16125 18978 22957 23809 18222
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The protein bands which migrated with a MW of 21 500 and larger protein standards were assumed to represent hemoglobin subunits to which POE was bound. This was con¢rmed by MALDI mass spectrometry analysis of PHP separated by SDS-PAGE and blotted onto nitrocellulose. Individual bands were cut from the nitrocellulose, dissolved in acetone, and their MW was determined by MALDI mass spectrometry. Samples of puri¢ed human hemoglobin were included as a standard for molecular weight analysis by mass spectrometry. Puri¢ed hemoglobin samples separated by this method contained a single band which co-migrated with the 14 400 MW protein standard. Separation of the K and L subunits was not observed on the 10% gels used for these experiments; however, MALDI-MS analysis of the band resolved two species with molecular weights of 15 324 and 16 090 (Table 1). These MWs corresponded to the expected values for K and L subunits, respectively. In the PHP samples, band 1 contained proteins with molecular weights of 15 422 and 16 125. Pyridoxal-5-phosphate has a MW of 247 and these molecular weights probably re£ect a mixture of pyridoxalated and unadulterated subunits. The second band on the gel (MW = 18 978) was considered to be representative of mixture of K and L subunits, some pyridoxalated, containing one POE molecule (MW = approximately 3100). Band 3 was considered to be representative of a mixture of K and L subunits, some pyridoxalated, containing two POE molecules. Band 4 was always found to be a much tighter band than other bands on the gel and consistently migrated slightly above band 3. MALDI analysis indicated that it had a molecular weight corresponding to a subunit containing two POEs. Based upon the large breadth of the signal observed from MALDI analysis (data not shown), it was expected that the signal obtained was from POE modi¢ed hemoglobin. The band was known to contain hemoglobin since band 4 was detected on Western blots probed with anti-human hemoglobin antibody (for instance, see Fig. 8A). The MALDI-MS spectrum for band 5 possessed a single peak corresponding to mass 18 222. This molecular weight could correspond to an K subunit containing a POE but no pyridoxal-5-phosphate. Alter-
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5 was in£uenced by the amount of PEG added to the reaction. Reactions which contained a 20-fold excess of PEG had signi¢cantly more material which migrated slowly on the gels and only a small amount of material which migrated equivalently to unmodi¢ed hemoglobin. In contrast, addition of equimolar amounts of hemoglobin and PEG resulted in an elution pro¢le where the majority of the material eluted equivalently to unmodi¢ed hemoglobin and relatively little material migrated equivalently to modi¢ed hemoglobin in PHP. In general, the SDS-PAGE pro¢le of the 20UPEG-modi¢ed PLP-Hb was similar (rela-
Fig. 4. SDS-PAGE analysis of PEG hemoglobin. SDS-PAGE analysis of PHP compared to pyridoxalated hemoglobins containing various degrees of modi¢cation with monomethoxyPEG. Lane 1 contains PHP. The ratio of PEG to hemoglobin in the modi¢cations were 20U (lane 2), 10U (lane 3), 5U (lane 4), 1U (lane 5). Lane 6 contains pyridoxalated hemoglobin. Hemoglobin crosslinked between L subunits with a 3400 MW PEG is shown in lane 7. Lane 8 contains stroma-free hemoglobin. Lane 9 contains molecular weight markers.
natively, if the signal were due to a doubly charged species, the actual molecular weight would correspond to two subunits crosslinked with a POE molecule. No signal was detected by MALDI-MS for any of the protein species above band 5. In order to further assess the properties of modi¢ed hemoglobin subunits derivatized with POE, additional modi¢ed hemoglobin species were prepared. In this case, pyridoxalated stroma-free hemoglobin (PLP-Hb) was modi¢ed with 3000 MW PEG which was mono-functional (one end contained N-hydroxysuccinimide and the other a monomethoxy group). A series of modi¢ed hemoglobins were made in which the PEG to hemoglobin ratio in the reactions was 1:1, 5:1, 10:1 or 20:1. SDS-PAGE analysis of these derivatives is shown in Fig. 4 where lane 1 contained PHP and lanes 2^5 contained hemoglobins modi¢ed at the PEG:hemoglobin ratios listed above (1U to 20U). Lane 6 contained PLP-Hb and lane 8 contained stroma-free hemoglobin for reference. The pro¢le of the modi¢ed hemoglobin shown in lanes 2^
Fig. 5. SEC analysis of PEG hemoglobin. Pyridoxalated hemoglobin was reacted with molar excess (1U, 5U, 10U, or 20U) of monofunctional PEG and analyzed by SEC. PHP displays the bimodal distribution described previously. 1UPEG hemoglobin elutes slowly, indicating only minor modi¢cation. 5UPEG hemoglobin approximately co-elutes with the major peak of PHP. 10U and 20UPEG hemoglobin elute slightly more rapidly than the major peak of PHP, but not as rapidly as the high MW component of PHP. The presence of a small percentage of high molecular weight material in the 20U reaction is believed to be due to contamination of the monomethoxy-PEG with some bi-functional reagent.
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tive migration distance of the bands and the number of bands) to the pro¢le observed for PHP up to band 5, with the exception of band 4 in PHP. No tight band was observed in this PEG hemoglobin preparation. However in PEG hemoglobin preparations made at 1U and 5UPEG:hemoglobin ratios, a tight band which co-eluted with carbonic anhydrase in stroma-free hemoglobin (Lane 8) was observed. Small di¡erences in the migration distance of the PEG modi¢ed species were attributed to di¡erences in the size and functionality between POE and PEG. A hemoglobin species which contained a speci¢c crosslink between the L-93 cysteine residues was used as a standard to con¢rm the elution position of a crosslinked L subunit. Lane 7 on the gel contained this species (XL-Hb). The unmodi¢ed K subunits migrated at the gel front with a mobility equivalent to the 14 400 MW standard. A species which was not crosslinked, but which was derivatized with a monofunctional form of the PEG at the L-93 cysteine migrated equivalently to a species in the PHP with subunits containing one POE chain (band 2). The crosslinked L subunits migrated equivalently to band 5 in PHP. However, a band containing multiple PEGs in the 20U and 10UPEG reactions also migrated in the same region of the gel. It is not possible to distinguish the multi-PEG modi¢ed subunits from the crosslinked species on these SDS-PAGE gels. PHP contained signi¢cantly more high MW species, based upon migration distance, than the PEG modi¢ed hemoglobin. These species were postulated to be crosslinked subunits which also contained additional POE molecules attached at only one position. In theory, the distinct PHP bands observed in SDS-PAGE should correspond to globin subunits derivatized with 1, 2, 3, 4, or 5 POEs (MW V19 000, 24 000, 27 000, 30 000, and 33 000, respectively) and globin dimers (MW V35 000). However, MALDI-TOF analysis of isolated bands as well as analysis of hemoglobin derivatized with monofunctional PEG demonstrated that PEG-modi¢ed proteins migrate aberrantly in SDS-PAGE. Other observations of anomalies in migration of PEG-proteins during SDS-PAGE have been reported. For example, IL-2 molecules with 2 and 3 PEGs co-migrated on gels [40]. Size exclusion analysis of PHP and the PEG hemoglobin illustrated the di¡erence between highly
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modi¢ed species and crosslinked species. Fig. 5 shows an overlay of chromatograms for PHP and the PEG-modi¢ed material. Even in derivatives which were synthesized with 10U or 20U excess of PEG to hemoglobin, the elution pro¢les were monomodal. The randomly PEG-modi¢ed hemoglobins were eluted from the SEC column after the population of high molecular weight species in PHP. Hemoglobin reacted with a 5U excess of PEG eluted in the same region of the chromatogram as the lower molecular weight peak of PHP. Based upon the elution pattern of the PEG hemoglobin, the lower MW peak of PHP was interpreted as hemoglobin tetramers with varying degrees of derivatization with POE. The higher MW portion of PHP was deduced to be tetramers joined to other tetramers with a POE crosslink. The SEC chromatogram shown in Fig. 5 illustrates a key di¡erence in the structure of proteins modi¢ed with a mono- vs bi-functional PEG molecule. Higher order tetramers are found in preparations made using a bi-functional reagent. If the chemical modi¢cation conditions are not tightly regulated, large networks of polymerized protein molecules can occur (leading to gelation of the proteins in some cases (data not shown)).
Fig. 6. IEF analysis of PHP. PHP focused as a broad band with an average pI of 5.8 on 3^9 pH gels. Unmodi¢ed hemoglobin focused as a tight band with a pI of approximately 7.1.
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Fig. 7. Western blots of PHP. Each of the panels shown contains a gel with a lane with (1) puri¢ed enzyme, (2) PHP and (3) molecular weight markers. (A) A Western blot probed with: anti-human catalase, (B) anti-human SOD, and (C) anti-human carbonic anhydrase. In each panel, the enzyme content of the PHP has been modi¢ed to some extent by derivatization with POE.
In addition to SDS-PAGE, PHP species were also examined by IEF analysis. PHP appeared as a broad band between protein standards of known isoelectric points (5.0^6.5) as shown in Fig. 6. The average isoelectric point of PHP was estimated to be 5.8. The pI of unmodi¢ed hemoglobin, which co-migrates with the hemoglobin contained in the standard protein mix, is approximately 7.1. No attempt to quantify any of the species on the Coomassie blue stained gels (SDS-PAGE or IEF) was made due to previous reports that PEG modi¢ed proteins were not stained quantitatively compared to their non-PEG-modi¢ed counterparts [40]. The hydrodynamic radii (RH ) of PHP and other hemoglobin solutions were determined by dynamic light scattering on a DynaPro Molecular Sizing Instrument. The puri¢ed hemoglobin generated a narrow monomodal distribution with a mean RH of 3.2 nm and a standard deviation of 0.5 nm. The average RH for ¢ve lots of PHP was 7.2 þ 0.6 nm. All ¢ve PHP samples had broad unimodal distributions with average standard deviation values of 2.1 nm. These molecular sizes for PHP and hemoglobin agree with
those reported by Vandegri¡ et al. (RG = 7.2 and 2.7 nm, respectively) [42] determined by measuring the colloidal osmotic pressure of the proteins in solution. PHP is a heterogeneous mixture of di¡erent forms of derivatized hemoglobin subunits which can be manufactured reproducibly and has been shown to be safe through pre-clinical and clinical trials. Based upon the manufacturing process, PHP was expected to contain many of the soluble enzymes which are found together with hemoglobin in red blood cells. The presence of some of these enzymes in PHP has been demonstrated by direct assay of their activities in PHP [43]. In addition, an immunoblot of PHP con¢rmed the presence of the enzymes catalase, SOD and carbonic anhydrase (Fig. 7). The enzymes were also derivatized with polyoxyethylene as evidenced by their increased molecular weight as compared to unmodi¢ed protein standards on the blots. The presence of enzymes, such as catalase, in PHP preparations may be a signi¢cant factor in controlling the oxidation rate of the heme groups [11,43] or may have bene¢cial e¡ects in treatment during certain disease states. Catalase and superoxide dismu-
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Fig. 8. Western blot of fractions from a¤nity chromatography. (A) An immunoblot probed with anti-human hemoglobin antibody. The load and unbound fractions have similar pro¢les, but the bound material is enriched for high molecular weight hemoglobin components. (B) An immunoblot probed with anti-human catalase antibody. Load, unbound and bound fractions possess similar pro¢les.
Fig. 9. PHP in solution. PHP is composed of tetrameric, pyridoxalated subunits bound to POE. A fraction of the tetramers are linked to other tetramers (and other red cell proteins) by POE, which is a bifunctional reagent capable of reacting with primary amines. The tetramers (of crosslinked and non-crosslinked species) are free to dissociate and re-associate depending on the solution characteristics.
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tase combinations have been evaluated for scavenging reactive oxygen species in animals [44]. The exogenous addition of superoxide dismutase and catalase to hemoglobin-based pharmaceuticals has been suggested for treatment of ischemia reperfusion injury and as a method to decrease hemoprotein-associated free radical events following oxidant challenge [45^ 48]. The enzymes were not only expected to be derivatized with POE, but were also expected to be attached to other proteins due to the bifunctional nature of POE. This was con¢rmed for catalase by examining the catalase-containing species in PHP. An a¤nity column was made with anti-catalase antibody, and PHP was applied to the column. The column was washed extensively with bu¡er to remove any non-speci¢cally bound material and the catalase containing material which was bound to the column was eluted with pH 2.5 glycine bu¡er. Samples of the load, non-bound (the £ow through) and bound fractions were separated in duplicate on a SDS-PAGE gel and blotted to nitrocellulose. One set of samples was probed with anti-hemoglobin antibody, while the other set was probed with anti-catalase antibody. The results are shown in Fig. 8. The blots probed with anti-catalase antibody (Fig. 8B) indicated that much catalase-containing material £owed through the column (non-bound fraction). However, catalase containing material was also speci¢cally bound and eluted with the low pH wash. The distribution of catalase species in load, unbound and bound fractions were all similar. The blots probed with antihemoglobin antibody (Fig. 8A), showed that the pro¢les of load and unbound fractions were similar, but that the bound material was enriched for higher molecular weight hemoglobin species. This high molecular weight hemoglobin represented material which was crosslinked to catalase by POE. The tethering of these potentially bene¢cial enzymes to hemoglobin would not occur if the conjugate were mono-functional PEG. Only a bifunctional molecule, such as the POE used here, will result in crosslinks between proteins. The crosslink ensures that these tethered enzymes are co-distributed with PHP within the body. However, at this time, the tissue distribution of the di¡erent species in PHP has not been elucidated. PHP is a complex mixture of di¡erent derivatives
of hemoglobin. Based upon the chemical characterization of PHP and the properties of the chemical modi¢cation agents, our current interpretation of the structure of PHP is shown in Fig. 9. The product primarily consists of modi¢ed tetrameric hemoglobin with a portion existing as crosslinked tetrameric species. Less than 1% of the tetramers are expected to be found as unmodi¢ed species. The tetrameric species can dissociate into dimers while the dimers can reassociate with other modi¢ed or unmodi¢ed dimers to reform tetramers. Many of the other soluble proteins found in the red blood cell are also found in PHP. These proteins are also modi¢ed by the attachment of POE and are likely to be crosslinked to PHP. The safety of PHP has been extensively studied in a number of animal models and is currently being evaluated in human clinical trials [28,49,50]. Acknowledgements We thank Dr. Dan Snyder of Protein Solutions for the dynamic light scattering measurements. Thanks also to Dr. Carol Haney and N. Srinivasan of The North Carolina State University for MALDI-MS analyses. We also thank Dr. Teresa Keng and Dr. Chris Privalle for critical review of the manuscript and Dr. Yuji Iwashita for helpful discussions about PHP.
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