Functional properties of a new crosslinked hemoglobin designed for use as a red cell substitute

A new crosslinking agent, bis-pyridoxal tetraphosphate, (bis-PL)P4, was used to prevent dissociation of the hemoglobin (Hb) tetramer. Yields in excess of 75 percent of intramolecularly crosslinked (bis-PL)P4Hb have been obtained using stoichiometric amounts of the crosslinking reagent. Some functional properties of (bis-PL)P4Hb have been evaluated in vitro and in vivo. Oxygen affinity was substantially reduced (p50 = 31 torr at 37 degrees C, pH 7.4, pCO2 = 40 torr), while the Bohr coefficient was -0.27 of H+ per mol of O2. Owing to its right-shifted dissociation curve, (bis-PL)P4Hb still yielded a p50 of 15 torr at a low temperature (16 degrees C), as compared with only 3 torr for normal adult Hb (HbA). Clearance of (bis-PL)P4Hb from plasma was significantly delayed (t1/2 = 171 min, at a dose of 0.2 g/kg of body weight compared with that of HbA (t1/2 = 54 min). Heart rate, mean arterial blood pressure, and respiration remained stable or returned to normal values within hours after bolus injection of the hemoglobin. The (bis-PL)P4Hb was not excreted in the urine, in contrast to HbA (21% of the total dose of HbA appeared in the urine within the first 2 hrs). These results show that the covalent beta-beta crosslink prevents the renal excretion of (bis-PL)P4Hb, thereby significantly prolonging vascular retention. These properties, together with an increased ability to unload O2, make (bis-PL)P4Hb a promising new candidate as a red cell substitute.