Binding of acellular, native and cross-linked human hemoglobins to haptoglobin: enhanced distribution and clearance in the rat.

It is well established that hemoglobin resulting from red cell lysis binds to haptoglobin in plasma to form a complex. The increased molecular size precludes its filtration by the kidneys, redirecting it toward hepatocellular entry. Chemically cross-linked hemoglobins are designed to be resistant to renal excretion, even in the absence of haptoglobin. The manner in which binding to haptoglobin influences the pharmacokinetics of acellular cross-linked and native hemoglobins was investigated after intravenous injection of radiolabeled native human hemoglobin and trimesyl-(Lys82)beta-(Lys82)beta cross-linked human hemoglobin, at trace doses, into rats. Under these conditions, there is sufficient plasma haptoglobin for binding with hemoglobin. In vitro binding assayed by size-exclusion chromatography for bound and free hemoglobin revealed that, at <8 muM hemoglobin, native human hemoglobin was completely bound to rat haptoglobin, whereas only approximately 30% of trimesyl-(Lys82)beta-(Lys82)beta cross-linked hemoglobin was bound. Plasma disappearance of low doses (0.31 mumol/kg) of native and cross-linked hemoglobins was monoexponential (half-life = 23 and 33 min, respectively). The volume of distribution (40 vs. 19 ml/kg) and plasma clearance (1.22 vs. 0.4 ml.min(-1).kg(-1)) were higher for native than for cross-linked hemoglobin. Native and cross-linked human hemoglobins were found primarily in the liver, and not in the kidney, heart, lung, or spleen, mostly as degradation products. These pharmacokinetic findings suggest that the binding of hemoglobin to haptoglobin enhances its hepatocellular entry, clearance, and distribution.

Rates of release of nitric oxide from HbSNO and internal electron transfer.

The discovery that hemoglobin (Hb) in erythrocytes contains a fraction of beta-Cys-93 thiols as the nitrosylated derivative (HbSNO) led to the suggestion that this species is involved in transporting and releasing nitric oxide, which is the signal for local vasodilation. The release of NO from HbSNO requires an electron transfer to facilitate release and to regenerate the cysteine thiol via one-electron reduction in the absence of added thiols. An alternative mechanism, which has received much attention, transfers the nitrosyl group to an external thiol, which in turn would have to be reduced. The observed first order rate constant for the spontaneous oxidation of the ferrous heme of deoxy HbSNO is 1.0 x 10(-4)s(-1) in the absence of thiols. Under the same conditions, native Hb is stable. The oxidation of HbSNO occurs with the same rate constant that can be derived for the rate reported for the formation of HbNO from HbSNO. These similarities suggest that both processes involve the same reaction: internal electron transfer and direct release of nitric oxide.