TIME-RESOLVED FLUORESCENCE OF HEMOGLOBIN SPECIES

We used time-resolved fluorescence in the pico- to nanosecond time ran ge to monitor the presence of tetramers, dimers and monomers in carbon monoxyhemoglobin (COHb) solutions and to investigate how their distrib utions change under different experimental conditions. Comparison of f luorescence lifetime computed from the atomic coordinates of COHb (Vas quez et al., 1996) with those experimentally measured allowed identifi cation of molecular species present in the hemoglobin solution. It was possible to observe modification of the distribution of tetramers, di mers, monomers and species with disordered hemes produced by different experimental conditions. Protein concentration affected the detectabl e lifetimes, indicating increasing amounts of dimers and monomers at l ow protein concentrations, while the amount of inverted hemes was not modified. Titration with up to 1 M NaCl modified only the extent of di ssociation of hemoglobin into dimers, without affecting heme inversion and monomer formation. Hyperbaric pressure increased the amounts of d imers and monomers. This is the first time that monomeric subunits of hemoglobin have been detected at neutral pH in the normal system.