FLUORESCENT EFFECTOR AS A PROBE OF THE ALLOSTERIC EQUILIBRIUM IN METHEMOGLOBIN

A fluorescent analogue of diphosphoglycerate (DPG), hydroxy-pyrenetris ulfonate (HPT),was used as a probe of the allosteric equilibrium of me themoglobin. Like DPG, HPT binds, one per tetramer, with a higher affi nity to deoxyHb than to oxyHb. Once bound, the HPT fluorescence is que nched by energy transfer to the hemes. HPT can thus serve as a probe o f the conformational state of the hemoglobin tetramer: a higher quench ing indicates a stronger binding and therefore, more of the deoxy conf ormation. 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 H PT under the same conditions, to correct for the inner filter effect. The high spin ferric ligands, such as water and F, showed less fluores cence (more of the deoxy state) than low spin cyano-metHb. The aquo-me tHb samples showed a reversion to the oxyHb conformation above pH 7, a s expected due to the acid-alkaline transition forming hydroxy-metHb. Effecters such as bezafibrate, which do not bind to the same site as D PG, show an increase in the deoxy-like characteristics. (C) 1998 Publi shed by Elsevier Science B.V.