Heterogeneous motions within human apohemoglobin

Conservation of the secondary and tertiary protein organization of human ap ohemoglobin was observed at temperatures ranging from 7 to 25 degrees C usi ng CD spectra in the far-UV (200-250 nm) and near-UV (250-300 nm) regions. The dynamics of apohemoglobin were probed using fluorescence quenching expe riments on the Trp residues and an extrinsic dye (ANS or bis-ANS) located i n the heme cavities. The long decay time of the dye emission (> 10 ns) reve als the dynamics of the protein matrix averaged over the whole molecule, Th e short decay time of the Trp residue emission (congruent to 3 ns) probes t he dynamics of their close vicinities. When the temperature rises from 10 t o 20 degrees C, the average intraproteic motions throughout the whole apohe moglobin matrix are greatly accelerated, whereas the hydrophobic protein re gions around the alpha 14, beta 15 and beta 37 Trp residues appear much les s animated. These dynamic differences between the behavior of the softer ma trix and the packed rigid regions containing the tryptophans could be one o f the requisites for apohemoglobin stability. We suspect that the highly ri gid tryptophan domains in human apohemoglobin are likely to be knots.