Fluorescent effector as a probe of the allosteric equilibrium in methemoglobin.
A fluorescent analogue of diphosphoglycerate (DPG), hydroxy-pyrenetrisulfonate (HPT), was used as a probe of the allosteric equilibrium of methemoglobin. Like DPG, HPT binds, one per tetramer, with a higher affinity to deoxyHb than to oxyHb. Once bound, the HPT fluorescence is quenched by energy transfer to the hemes. HPT can thus serve as a probe of the conformational state of the hemoglobin tetramer: a higher quenching indicates a stronger binding and therefore, more of the deoxy conformation. Since HPT binds to the same site as DPG, it can be displaced by DPG in order to determine the fluorescence intensity of the free HPT under the same conditions, to correct for the inner filter effect. The high spin ferric ligands, such as water and F, showed less fluorescence (more of the deoxy state) than low spin cyano-metHb. The aquo-metHb samples showed a reversion to the oxyHb conformation above pH 7, as expected due to the acid-alkaline transition forming hydroxy-metHb. Effectors such as bezafibrate, which do not bind to the same site as DPG, show an increase in the deoxy-like characteristics.
Serbanescu
R
R
University of Bucarest, Department of Biophysics, Magurele, Romania.
Kiger
L
L
Poyart
C
C
Marden
M C
MC
D000495:Allosteric Site; D001190:Arylsulfonates / Q000378:metabolism*; D001629:Bezafibrate / Q000378:metabolism; D001667:Binding, Competitive; D004163:Diphosphoglyceric Acids / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008706:Methemoglobin / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013696:Temperature
D001190:Arylsulfonates; D004163:Diphosphoglyceric Acids; D005456:Fluorescent Dyes; D006454:Hemoglobins; D006418:Heme; C100003:deoxyhemoglobin; D008706:Methemoglobin; C005047:pyranine; D001629:Bezafibrate