Rate of nitric oxide scavenging by hemoglobin bound to haptoglobin

Abstract

Cell-free hemoglobin, released from the red cell, may play a major role in regulating the bioavailability of nitric oxide. The abundant serum protein haptoglobin, rapidly binds to free hemoglobin forming a stable complex accelerating its clearance. The haptoglobin gene is polymorphic with two classes of alleles denoted 1 and 2. We have previously demonstrated that the haptoglobin 1 protein-hemoglobin complex is cleared twice as fast as the haptoglobin 2 protein-hemoglobin complex. In this report, we explored whether haptoglobin binding to hemoglobin reduces the rate of nitric oxide scavenging using time-resolved absorption spectroscopy. We found that both the haptoglobin 1 and haptoglobin 2 protein complexes react with nitric oxide at the same rate as unbound cell-free hemoglobin. To confirm these results we developed a novel assay where free hemoglobin and hemoglobin bound to haptoglobin competed in the reaction with NO. The relative rate of the NO reaction was then determined by examining the amount of reacted species using analytical ultracentrifugation. Since complexation of hemoglobin with haptoglobin does not reduce NO scavenging, we propose that the haptoglobin genotype may influence nitric oxide bioavailability by determining the clearance rate of the haptoglobin-hemoglobin complex. We provide computer simulations showing that a twofold difference in the rate of uptake of the haptoglobin-hemoglobin complex by macrophages significantly affects nitric oxide bioavailability thereby providing a plausible explanation for why there is more vasospasm after subarachnoid hemorrhage in individuals and transgenic mice homozygous for the Hp 2 allele.

Figure 1

Sedimentation studies showing no unbound Hb in photlysis studies. (A) Raw absorption data from the analytical centrifuge taken at 415 nm, 2.5 hours after beginning the spin at 45,000 rpm. Each curve shows the absorbance due to Hb either free or bound to Hp plotted against position in a separate centrifuge tube. The radial positions of free Hb and Hb-Hp 2-2 have been shifted to line up their menisci with that of Hb-Hp 1-1. The Hb bound to the Hp 2-2 has completely sedimented at the time these data were collected and there is no evidence of free Hb in that sample. The sample containing Hp 1-1 has not completely sedimented but comparison to the free Hb sample indicates that there is little to no free Hb in the Hb-Hp 1-1 sample. (B) Species distributions were calculated using DCDT+ (version 6.31) software (J. Philo, Thousand Oaks, CA) [21].

Figure 2

Photolysis studies of Hb free and bound to either Hp 1-1 or Hp 2-2. Caged NO (2 mM) was mixed with Hb or Hb-Hp and released using a 355 nm wavelength laser pulse. The absorption was followed using a continuous probe beam focused onto a Streak camera. (A–C) Absorbance (after noise reduction using singular value decomposition) is plotted against wavelength following NO release for (A) unbound Hb, (B), Hb bound to Hp 1-1, and (C) Hb bound to Hp 2-2. (D–F) Initial and final species obtained by global analysis of data shown in the left hand column. The plots contain the initial species obtained from global analysis (OxyHb) and the final species (MetHb). The insets show the absorbance over time at 406 nm. (D) unbound Hb, (E), Hb bound to Hp 1-1, and (F) Hb bound to Hp 2-2.

Figure 3

Competition Experiments. (A) Absorption data collected about 141 minutes after the beginning of sedimentation. The absorbance from two consecutive scans along a centrifuge cell are shown. (B) The average relative bimolecular rate constants for the NO dioxygenation reaction. The average value of the relative rate constant of the reaction for free Hb (k_f) divided by that bound to Hp (k_b) is shown ± one standard deviation for Hp 1-1 and Hp 2-2 (from a total of about 5000 calculations from 4 separate experiments on each Hp).

Figure 4

Effect of cell-free Hb in the lumen and interstitial space on [NO]. Concentrations of NO in the presence of cell-free Hb with and without extravasation are plotted versus the distance along the vessel axis. The simulations were performed with 50% Hct (total RBC encapsulated Hb in the lumen was 10 mM) and red blood cell permeability of 450 µm s⁻¹. The position of the endothelium is indicated by the black arrow at 0.05 mm (peak NO availability). Cell-free Hb concentrations ([Hbcf]) were set to 2 and 4 µM and the concentrations of extravasated cell-free Hb into the interstitial space ([Hbextra]) were varied from 0, 0.5, and 1 µM. The figure shows that the concentration of NO at the endothelium was between about 0.11 µM and 0.18 µM for the different simulations shown. NO concentration increases when the cell-free Hb and extravasated cell-free Hb concentrations are lowered by a factor of two corresponding to the difference in Hb uptake rates by different Hp types.