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

Biochem J. 2004 Aug 15;382(Pt 1):183-9. doi: 10.1042/BJ20040156.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Hemoglobins / chemistry
  • Hemoglobins / metabolism*
  • Humans
  • Maleimides / chemistry
  • Maleimides / metabolism*
  • Nitric Oxide / chemistry
  • Nitric Oxide / metabolism*
  • Oxygen / chemistry
  • Oxygen / metabolism*
  • Polyethylene Glycols / chemistry
  • Polyethylene Glycols / metabolism*


  • Hemoglobins
  • Maleimides
  • maleimide-polyethylene glycol-modified hemoglobin, MP4
  • Nitric Oxide
  • Polyethylene Glycols
  • Oxygen