Oxidation and haem loss kinetics of poly(ethylene glycol)-conjugated haemoglobin (MP4): dissociation between in vitro and in vivo oxidation rates

Abstract

Haemoglobin-based oxygen carriers can undergo oxidation of ferrous haemoglobin into a non-functional ferric form with enhanced rates of haem loss. A recently developed human haemoglobin conjugated to maleimide-activated poly(ethylene glycol), termed MP4, has unique physicochemical properties (increased molecular radius, high oxygen affinity and low cooperativity) and lacks the typical hypertensive response observed with most cell-free haemoglobin solutions. The rate of in vitro MP4 autoxidation is higher compared with the rate for unmodified SFHb (stroma-free haemoglobin), both at room temperature (20-22 degrees C) and at 37 degrees C (P<0.001). This appears to be attributable to residual catalase activity in SFHb but not MP4. In contrast, MP4 and SFHb showed the same susceptibility to oxidation by reactive oxygen species generated by a xanthine-xanthine oxidase system. Once fully oxidized to methaemoglobin, the rate of in vitro haem loss was five times higher in MP4 compared with SFHb in the fast phase, which we assign to the beta subunits, whereas the slow phase (i.e. haem loss from alpha chains) showed similar rates for the two haemoglobins. Formation of MP4 methaemoglobin in vivo following transfusion in rats and humans was slower than predicted by its first-order in vitro autoxidation rate, and there was no appreciable accumulation of MP4 methaemoglobin in plasma before disappearing from the circulation. These results show that MP4 oxidation and haem loss characteristics observed in vitro provide information regarding the effect of poly(ethylene glycol) conjugation on the stability of the haemoglobin molecule, but do not correspond to the oxidation behaviour of MP4 in vivo.

Figure 1. Autoxidation of SFH and MP4

Upper panel: autoxidation of SFHb and MP4 at room temperature (20–22 °C; left panel) and 37 °C (right panel). Experiments were performed with four different preparations of each Hb type with a Hb concentration of 0.06 mM in PBS with 0.1 mM EDTA (pH 7.3). P<0.0001 (as determined by ANOVA) for both temperatures. *P<0.001 compared with SFHb (determined by the Bonferroni multiple comparison test). Lower panel: a Van't Hoff plot of the data shown in the upper panel, with the k_ox reported as a function of 1/°K. The inset shows the slopes of the regression lines separately for SFHb and MP4. *P<0.001 compared with SFHb (Student's t test).

Figure 2. Rates of autoxidation of SFH and MP4 in the presence and absence of catalase

The left panel (A) shows the inhibition of catalase activity by KCN. pO₂ was measured in an anaerobic cell containing 2 ml PBS and 0.1 ml catalase (final nominal activity 1000 units/ml). The reaction was started by adding 5 μl of 3% H₂O₂ (arrow) and pO₂ was monitored for an additional 60 s at 37 °C. The reaction was performed at KCN concentrations of 0, 20 and 50 mM. The right panel (B) shows the autoxidation of SFHb and MP4 in the presence of PBS only (■), 1000 units/ml catalase (△) and 50 mM KCN (●). Best fit lines were obtained as explained in the text, and Table 1 reports the values of the rate constants.

Figure 3. Hb oxidation by the xanthine/xanthine oxidase system

The upper panel shows a typical spectral change observed when 0.1 mM Hb in 50 mM potassium phosphate, 0.5 mM xanthine, 50 mM KCN and 1 mM EDTA (pH 7.3) is exposed to 0.5 units of xanthine oxidase at 37 °C. Spectra were obtained every 5 s. The lower panel shows the absorbance changes recorded at λ=576 nm to measure the maximal oxidation rate. The Figure also shows the decay of pO₂ (right axis) when the mixture is reacted in the absence of Hb. The maximal oxidation rate of Hb corresponds to the linear portion of the fall in pO₂.

Figure 4. Time course of haem loss from SFHb, MP4 and P5K2 MetHbs

Averaged data were fitted using a double-exponential decay (see text). The rates are reported in Table 2. n=3 per group.

Figure 5. Effect of 0.1 mM ascorbate on autoxidation of SFHb and MP4 to MetHb in PBS at 37 °C over 3 h

P<0.001 for both (as determined by ANOVA). *P=0.02 compared with PBS (as determined by Bonferroni's multiple comparison test).

Figure 6. Disappearance of plasma Hb and formation of MetHb

Left panel: rats exchange-transfused with MP4 (n=4). Right panel: humans transfused with different doses of MP4 (n=4 in each group). Humans received 200, 400 or 600 ml, and data were normalized for body weight. Average and S.E.M. of concentrations (g/l) are shown. The plasma Hb disappearance curves with 95% confidence limits were fitted using a single-exponential decay function. Note different scales.

Figure 7. Relationship between autoxidation rates and p50 or oxygen dissociation rate constants

Upper panel: relationship between the apparent k_ox and Hb–O₂ affinity (p50, i.e. the pO₂ at which half of Hb is saturated with oxygen) in various Hb types from the present study and from the literature [7]. The line represents the best linear fit when the point referring to MP4 is excluded from the calculation, with the 95% confidence limits, slope=(0.808±0.085)×10⁻³, intercept=(1.46±2.928)×10⁻³, P=0.0007. Lower panel: relationship between k_ox and the rate constant of oxygen dissociation from OxyHb in various Hb types from the literature [34,37]. The line represents the best linear fit when considering all data points, with the 95% confidence limits, slope=(0.648±0.141)×10⁻³, intercept=(−2.807±4.331)×10⁻³, P=0.04.