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.