MOLECULAR-DYNAMICS OF HEMOGLOBIN SUBUNITS AS SEEN BY FLUORESCENCE SPECTROSCOPY

Fluorescent conjugates of [human] .beta.A subunits and their respective heme-free derivatives were prepared in which a 1,5-N-iodoacetylaminoethyl-5-naphthylamine-1-sulfonate probe was specifically placed at the .beta.-93 or .beta.-112 cysteine. The fluorescence anisotropy decay and static fluorescence polarization of these conjugates were examined. Fluorescence measurements were also made using 1-anilino-8-naphthalenesulfonate complexes and the intrinsic fluorescence of the tryptophan groups. For the cases of the .beta.-93 and .beta.-112 conjugates there is substantial evidence for internal rotational freedom of the subunits. The internal mobility of the polypeptide is especially pronounced for the .beta.-112 conjugate. The 1-anilino-8-naphthalenesulfonate probe placed within the heme pocket shows no indication of any rotation other than that associated with the entire .beta.-subunit. Tryptophan fluorescence was measured for the apo-.beta. subunits and for the peptides .beta.(1-55) from Hb A and S. Perrin-Weber plots show the presence of multiple rotational modes suggesting mobility of the tryptophan groups.