Stability of a Potential Blood Substitute, HbXL99α, Under High Pressure

One important criteria for a plasma circulating hemoglobin blood substitute is resistance to subunit dissociation. For this reason, cross-linked hemoglobins (with low oxygen affinities) are being specifically designed to serve as potential blood substitutes. An example is HbXL99α, cross-linked between the α-subunits [PNAS (1987) 84:7280]. In the study presented here, the effects of up to 2 kilobars of pressure on the intrinsic fluorescence of HbXL99α, HbA and myoglobin were compared. Hemoglobin solutions were studied between 0.01 - 0.1g% in potassium phosphate or Hepes buffers, pH 7.4. Results show HbA exhibits a decrease in fluorescence intensity as a function of pressure. In contrast, HbXL99α as well myoglobin (a monomer) show essentially no significant intrinsic fluorescence changes as a function of pressure. These results suggest that HbXL99α is stable as a tetramer up to ^∼2 kilobars of pressure. In addition, high pressure intrinsic fluorescence studies provide a suitable technique for determining the subunit stability of hemoglobins.