Approaches to the engineering of hemoglobin based oxygen carriers

Molecular biology offers the opportunity to construct hemoglobin molecules as blood substitutes tailored to specific therapeutic applications. Oxygen affinity can be manipulated by amino acid substitutions in the heme pocket or on the protein surface. A response to the concentration of plasma-Cl- that mimics the effect of 2,3-DPG was also introduced in human Hb. Polymerization of tetrameric Hb prevents the vasoconstriction associated with infusion of Hb solutions. Polymers have been obtained through the formation of intermolecular S-S bonds between cysteine residues introduced on the Hb surface. In a mouse model, transfusion of polymeric hemoglobins reduced the volume of cerebral infarction. This effect was particularly evident with polymers having a high oxygen affinity (P50≤˜2.0 Torr) and no cooperativity (n = 1). These are the same functional characteristics of myoglobin. Polymers of myoglobin have been constructed and could be a viable alternative to the use of polymeric hemoglobins in some pathological conditions

Effect of disordered hemes on energy transfer rates between tryptophans and heme in myoglobin

Our recent linear dichroism study of heme transitions (Gryczynski, Z., E. Bucci, and J. Kusba. 1993. Photochem. Photobiology. in press) indicate that heme cannot be considered a planar oscillator when it acts as an acceptor of radiationless excitation energy transfer from tryptophan. The linear nature of the heme absorption transition moment in the near-UV region implies a strong dependence of the transfer rate factors on the relative angular position of the heme and tryptophan, i.e., on the kappa 2 orientation parameter of the Förster equation. Using the atomic coordinates of SW myoglobin we have estimated the variation of kappa 2 parameter as a function of the heme absorption transition moment direction. The simulations proved that transfer is very efficient and anticipates lifetimes in the picosecond range. Also, they showed that transfer is very sensitive to rotations of the heme around its alpha-gamma-meso-axis, which may reduce the efficiency of transfer to almost zero values, producing lifetimes very similar to those of free tryptophan, in the nanosecond range. Comparisons between the lifetime values reported in the literature and those here estimated suggest that natural heme disorder, in which heme is rotated 180 degrees around its meso axis, is at the origin of the nanosecond lifetimes found in myoglobin systems