Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology
Chemical characterization of pyridoxalated hemoglobin polyoxyethylene conjugate
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], [3], [4], [5]. The crystal structures of a number of different 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 α and 2 β subunits. Stable dimers consisting of an α and β 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 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], [10], [11]. These insults are opposed by antioxidant enzymes and reductases [12], [13], which efficiently 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], [16], [17], [18]. Hemoglobin therapeutics have been designed as oxygen-carrying fluids 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 modification due to renal toxicity, as described by Savitsky et al. [21]. In addition, the plasma half-life of unmodified hemoglobin is short and the oxygen affinity of hemoglobin in plasma may be too great to allow efficient delivery of oxygen in those applications where oxygen delivery is desired [22]. These limitations to the use of unmodified human hemoglobin have been circumvented by chemical modification of the hemoglobin in one or more of the following ways: (1) crosslinking α, β dimers; (2) polymerizing hemoglobin; or (3) binding polymers to the surface of hemoglobin [23], [24], [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 affinity [29]. The pyridoxalated stroma-free hemoglobin is then modified with the homo-bifunctional molecule POE to increase the hydrodynamic volume (apparent molecular weight) of the molecule. The resulting modified hemoglobin entity is purified 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], [31], [32], [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-modified 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 modification 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 modification agent used during production of PHP.
Section snippets
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 modified 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 α,α-crosslinked hemoglobin was a gift from the Walter Reed Army Institute of Research.
In addition to PHP, several hemoglobin
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±3385, the number average MW was determined to be 105 122±3566 and the weight average MW was determined to be 187 000±8300. The profile of the chromatogram indicated that the MW distribution was bimodal as shown in Fig. 1. The polydispersity of the PHP was 1.778±0.031. Typical peak
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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