Abstract
The relative intrinsic and extrinsic fluorescence of hemoglobins are established markers of the R (oxy) → T (deoxy) transition and reveal site-specific conformational differences amongst hemoglobin (Hb) mutants. The Hb central cavity has been probed by binding the fluorescent analogue of 2,3-diphosphoglycerate, 8-hydroxy-1,3,6-pyrenetrisulfonate (HPT). An approach to quantify binding of HPT to HbC (β6 Lys) and HbA (β6 Glu), by steady-state front-face fluorescence spectroscopy using both the intrinsic and extrinsic emissions, is presented. When HPT specifically binds to Hb, efficient fluorescence energy transfer from the intrinsic fluorescence of Hb to HPT occurs, decreasing the intrinsic fluorescence of Hb that plateaus upon stoichiometric binding. HPT fluorescence is significantly but not totally quenched upon binding to HbA and HbC. HPT exhibits a molecular binding ratio of 2:1 to HbC, in contrast to HbA (1:1 binding). The apparent secondary binding site for HbC is weaker (KD1 = 25 μM vs. KD2 = 0.15 mM). Conformational alterations of HbC at the αα and ββ clefts of the central cavity are further supported by these data.
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