Librational modes in liganded and unliganded hemoglobin as seen by fluorescence spectroscopy

Hemoglobin labeled with N-iodoacetylaminoethyl-5-naphthalene-1-sulfonate at the beta-93 cysteine shows normal allosteric properties including the Bohr effect and heme-heme-interaction, and the oxygen affinity is somewhat increased. Viscosity-resolved correlation times obtained from direct measurement of the decay of fluorescence anisotropy show values of 4.15 +/- 0.23 and 9.13 +/- 0.46 ns for the liganded and unliganded derivatives, respectively, in 0.05 M tris buffer, pH 7.5 at 15 degrees C. Addition of 1 mM inositol hexaphosphate to the liganded and unliganded derivatives results in correlation times of 9.31 +/- 0.87 and 43.69 +/- 16.04 ns, respectively. Similar values of correlation times are obtained from Perrin plots. Considering that the correlation times expected for the single subunits, the dimers, and the tetramers of the system are approximately 10-15, 20-30, and above 40 ns, respectively, it can be concluded that the molecule of hemoglobin becomes increasingly rigid upon removal of ligands and addition of inositol hexaphosphate. These observations support the hypothesis of a functional relevance of the internal flexibility of hemoglobin.