Chemical characterization of pyridoxalated hemoglobin polyoxyethylene conjugate

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¿ omitted¿000 and a weight average molecular weight of approximately 187¿ omitted¿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.