Protein Engineering, Vol. 14, No. 5, 359-366,
May 2001
© 2001 Oxford University Press
Activation of the low oxygen affinity-inducing potential of the Asn108(ß)
Lys mutation of Hb-Presbyterian on intramolecular 
-fumaryl cross-bridging
1 Department of Physiology and Biophysics and 3 Division of Hematology, Albert Einstein College of Medicine, Bronx, NY 10461 and 4 Structural BioInformatics, San Diego, CA 92127, USA
The Asn108ß
Lys mutation in hemoglobin (HbPresbyterian mutation) endows a low O2 affinity-inducing propensity to the protein. Introduction of a fumaryl cross-bridge between its two 
99 lysine residues also induces a low O2 affinity into HbA. We have now engineered an -fumaryl cross-bridge into Hb-Presbyterian to determine the synergy or additivity, if any, that can be achieved between these two low O2 affinity-inducing structural perturbations. Despite the presence of the additional 
-amino group of Lys108(ß) within the central cavity, the -amino group of Lys99() of deoxy Hb-Presbyterian retained high selectivity for -fumaryl cross-bridging, with an overall efficiency comparable to that with HbA. The -fumaryl cross-linking of Hb-Presbyterian reduced its O2 affinity much more significantly than that observed with HbA, indicating a synergy between the two low O2 affinity-inducing structural perturbations. Apparently, the -fumaryl cross-bridge in Hb-Presbyterian activates part of the latent low O2 affinity-inducing potential of Lys108(ß) that is generally activated in the presence of chloride. The synergy between the Asn108(ß)Lys mutation and the -fumaryl cross-bridging was conserved in the presence of chloride, but not in the presence of DPG. Furthermore, in the presence of chloride and DPG, -fumaryl Hb-Presbyterian accessed a low O2 affinity T-state that is accessed by HbA, -HbA and Hb-Presbyterian only in the presence of IHP. Isoelectric focusing analysis suggested that the -fumaryl cross-linking of Hb-Presbyterian induces changes in the ionization behavior of one or more of the functional groups neighboring Lys99() and Lys108(ß) [presumably His103() and/or Glu101(ß)] to compensate for the extra positive charge of Lys108(ß). Molecular modeling studies identified two potential chloride binding sites per ß dimer within the middle of the central cavity of -fumaryl HbA involving residues His103(), Arg104(ß) and Asn108(ß). The affinity of these sites is increased in -fumaryl Hb-Presbyterian as a result of the Asn108(ß)Lys mutation. Thus, the results of the present study suggest that the enhanced neutralization of the positive charges in the middle of the central cavity of Hb achieved by these two electrostatic modifications, one (the -fumaryl cross-bridge) acting directly and the other (the Presbyterian mutation) acting indirectly through the mediation of chloride ion binding, facilitates the - fumaryl-Hb Presbyterian to access a low O2 affinity T-state structure much more readily than either Hb-Presbyterian or -fumaryl HbA.