Kinetics of NO and O2 binding to a maleimide poly(ethylene glycol)-conjugated human haemoglobin

Abstract

The hypertensive effect observed with most cell-free haemoglobins has been proposed to result from NO scavenging. However, a newly developed PEG [poly(ethylene glycol)]-conjugated haemoglobin, MalPEG-Hb [maleimide-activated PEG-conjugated haemoglobin], is non-hypertensive with unique physicochemical properties: high O2 affinity, low co-operativity and large molecular radius. It is therefore of interest to compare the ligand-binding properties of MalPEG-Hb with unmodified cell-free HbA (stroma-free human haemoglobin). NO association rates for deoxy and oxyMalPEG-Hb and HbA were found to be identical. These results confirm the lack of correlation between hypertension and NO for a similar modified haemoglobin with high molecular radius and low p50 (pO2 at which haemoglobin is half-saturated with O2) [Rohlfs, Bruner, Chiu, Gonzales, Gonzales, Magde, Magde, Vandegriff and Winslow (1998) J. Biol. Chem. 273, 12128-12134]. The R-state O2 association kinetic constants were also the same for the two haemoglobins. However, even though the p50 of MalPEG-Hb is approx. half of that of HbA, the biphasic O2 dissociation rates measured at relatively high pO2 (150 Torr) were 2-fold higher, giving rise to a 2-fold lower R-state equilibrium association constant for MalPEG-Hb compared with HbA. Thus the O2 affinity of MalPEG-Hb is higher only at pO2 values lower than the intersection point of the O2 equilibrium curves for MalPEG-Hb and HbA. In summary, the present studies found similar rates of NO binding to HbA and MalPEG-Hb, eliminating the possibility that the lack of vasoactivity of MalPEG-Hb is simply the result of reduced molecular reactivity with NO. Alternatively, the unique O2-binding characteristics with low p50 and co-operativity suggest that the 'R-state' conformation of MalPEG-Hb is in a more T-state configuration and restricted from conformational change.

Figures

Figure 1. Time courses of NO association to deoxyhaemoglobin

Symbols show data points measured at 436 nm. Experimental conditions: 0.1 M Hepes buffer containing 0.1 M NaCl (pH 7.4) and temperature 23 °C. Solid lines are single-exponential fits to the time courses, assuming pseudo-first-order reaction conditions. Zero time is set to 3 ms to account for the dead time of the rapid-mixing apparatus. The time courses were normalized to give the same amplitude. The bimolecular association rate, k′, is calculated using the pseudo-first-order approximation in NO: k′=kobs/[NO]. The inset shows the same data on a semi-logarithmic plot; the straight lines indicate that the pseudo-first-order hypothesis holds true.

Figure 2. Time courses of NO oxidation of oxyhaemoglobin

Symbols show data points measured at 419 nm. Solid lines are single-exponential fits to the time courses, assuming pseudo-first-order reaction conditions. Zero time is set to 3 ms to account for the dead time of the rapid-mixing apparatus. The time courses were normalized to give the same amplitude. Experimental conditions were as described in Figure 1. The bimolecular oxidation rate, kox, is calculated using the pseudo-first-order approximation in NO: kox=kobs/[NO]. The inset details are same as described in Figure 1.

Figure 3. Time courses of O2 association to deoxyhaemoglobins following laser flash photolysis (hν)

Symbols show data points measured at 436 nm. Experimental conditions were as described in Figure 1. The photochemical yield at the beginning of the bimolecular recombination of O2 was 5–10% due to the low photochemical yield of HbO2 and the geminate recombination occurring on a much faster time scale than that reported in the Figure. Solid lines are double-exponential fits to the time courses, forcing equal amplitudes for the two phases according to eqn (1) to obtain pseudo-first-order rates for β [fast phase (f)] and α [slow phase (s)] subunits using kβ,α=kobs(f,s)/[O2].

Figure 4. Time courses of O2 dissociation from oxyhaemoglobin maintained in the R-state conformation by saturation with CO

Symbols show data points measured at 412 nm. Experimental conditions were as described in Figure 1. Solid lines are fits to the time courses. Zero time is set to 3 ms to account for the dead time of the rapid-mixing apparatus. The time courses were fitted to the double-exponential expression in eqn (1).

Figure 5. Oxygen equilibrium curves for HbA and MalPEG-Hb measured in 0.1 M Hepes, 0.1 M Cl−, 0.1 M EDTA and catalase at pH 7.4 and 23 °C

The experiment was performed by enzymic deoxygenation as described previously [26]. The fitted curves were determined by Adair analysis as described previously [34].