Fluorescent derivatives of human hemoglobin. Differences in interaction of the porphyrin with the protein between the alpha and beta subunits.
The fluorescence properties of porphyrin were compared in protoporphyrin-protoheme hybrid hemoglobins (Hb) in which the protohemes (Fe) in either the alpha or beta subunits were substituted with protoporphyrin IX, alpha (P)2 beta (Fe)2-Hb and alpha (Fe)2 beta(P)2-Hb, respectively. The fluorescence lifetimes of the porphyrin in these hybrid derivatives are shorter than for the tetra-substituted protoporphyrin Hb, indicating that there is energy transfer from the porphyrin to the heme. The energy transfer resulted in only approximately 20% diminution of donor fluorescence, thereby allowing for the examination by fluorescence of the interaction of porphyrin with a given subunit peptide chain. Fluorescence line-narrowing spectroscopy, a high resolution fluorescence technique that involves laser excitation, produced vibrationally resolved emission spectra in which the zero phonon lines were separated from a broadened background consisting of phonon wings. Resolved spectra were obtained in the temperature range of 5-50 K; above 70 K the spectra were unresolved. By changing the excitation frequency the mean energy of the 0,0 transition, characterizing the separation in energy between the ground and excited states, can be determined. For both derivatives a bimodal distribution was seen, indicating that more than one conformation of molecules was present. The distribution of 0,0 transition energies of the porphyrin in the beta-chain in alpha(Fe)2 beta(P)2-Hb was shifted to lower energy relative to the alpha chain in alpha(P)2 beta(P)2-Hb, whereas the excited state molecules in the two subunits had very little difference in vibrational energy. These data point to the same configuration of the porphyrins in the two sites, but differences in the electric field for the alpha and beta subunits.
Sudhakar
K
K
Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia 19104.
Loe
S
S
Yonetani
T
T
Vanderkooi
J M
JM
D006418:Heme / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D011166:Porphyrins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013696:Temperature
D006454:Hemoglobins; D011166:Porphyrins; D006418:Heme