Probing the Hemoglobin Central Cavity by Direct Quantification of Effector Binding Using Fluorescence Lifetime Methods
David S. Gottfried, Laura J. Juszczak, Nazim A. Fataliev, A. Seetharama Acharya, Rhoda Elison Hirsch, Joel M. Friedman
1997
Journal of Biological Chemistry
Time-resolved fluorescence methods have been used to show that 8-hydroxy-1,3,6-pyrenetrisulfonate (HPT), a fluorescent analog of 2,3-diphosphoglycerate, binds to the central cavity of carboxyhemoglobin A (HbACO) at pH 6.35. A direct quantitative approach, based on the distinctive free and bound HPT fluorescent lifetimes of 5.6 ns and ϳ27 ps, respectively, was developed to measure the binding affinity of this probe. HPT binds to a single site and is displaced by inositol hexaphosphate at a 1:1
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... l ratio, indicating that binding occurs at the 2,3-diphosphoglycerate site in the central cavity. Furthermore, the results imply that low pH HbACO exists as an altered R state and not an equilibrium mixture of R and T states. The probe was also used to monitor competitive effector binding and to compare the affinity of the binding site in several cross-bridged HbA derivatives. The solvent-accessible central cavity of hemoglobin A (HbA) 1 contains functionally important binding sites for several classes of allosteric effectors that facilitate the lowering of oxygen affinity (1, 2). The -subunit end of the central cavity contains a cluster of eight positive charges that interact with the negative charges of 2,3-diphosphoglycerate (DPG) (3). This site also binds a variety of other negatively charged effectors such as inositol hexaphosphate (IHP), inorganic phosphate, chloride, and polyglutamic acid. The other end (␣-subunit) of the central cavity contains additional binding sites, particularly for chloride ions. Another class of potent effectors derived from clofibric acid and bezafibrate (e.g. L35) bind near the middle of the central cavity with their negative charges projecting toward the ␣-subunit end (4 -6). Binding of these effectors is also associated with a reduction in oxygen affinity. Study of these effectors is of practical interest since control of oxygen affinity is a necessary component for the design of acellular Hb-based oxygen carriers (7, 8). X-ray crystallographic studies are important both in pinpointing effector-binding sites and in characterizing the geometry of the effector-bound site (2). However, other methods must be used to determine the structural and functional interactions that are important in solution and to perform titration studies for obtaining binding constants as a function of solution conditions and/or structural state. Functionally relevant synergistic and antagonistic effects among effectors are also best elucidated through solution studies. Functional characterization of hemoglobin suggests that synergistic and competitive activity can occur when combinations of effectors are bound (4). Of the various allosteric effectors, the interactions of DPG (the natural allosteric effector found in the red blood cell) and its analogs in HbA have been investigated the most extensively. However, the binding of DPG can only be measured indirectly through its effects on ligand reactivity and on spectroscopically accessible chromophores such as the heme groups. In this report, we present an extension of the use of a fluorescent analog of DPG to probe directly the hemoglobin DPG-binding site in a quantitative fashion. Gibson and MacQuarrie (9, 10) showed that 8-hydroxy-1,3,6pyrenetrisulfonate (HPT) can be used as a fluorescent analog of DPG. Utilizing the observation that the fluorescence signal from HPT is highly quenched when bound to HbA, they performed steady-state fluorescence intensity measurements and established that HPT has a lower affinity for the DPG-binding site than DPG or IHP. A subsequent study by that group (11), using HPT as a probe of the DPG-binding site, focused on the detection of ligand-binding intermediates occurring along the R to T transition pathway. In those initial experiments, the results were limited in large part by two aspects of the methodology. (i) To monitor changes in HPT fluorescence in the presence of the highly absorbing heme, low concentrations of Hb had to be used. As a result, dissociation of the tetramer produced ␣-dimers in a concentration high enough to complicate data analysis. (ii) The use of steady-state fluorescence quenching as a means of obtaining titration data does not allow for probing of the HPT-bound species or for a direct determination of the fractions of both bound and unbound effector in a single measurement. In the present study, these limitations were overcome using a combination of front-face optical geometry and fluorescence lifetime measurements. The use of front-face excitation allows for the observation of HPT fluorescence upon binding to hemoglobin at high concentrations without innerfilter effects or substantial dimer formation. Front-face fluorescence techniques have been used previously to probe the highly quenched fluorescence from tryptophans within both hemoglobins and myoglobins (12) . Fluorescence lifetime measurements of HPT in the presence of HbA reveal two distinct and easily resolved lifetime components that allow unambiguous determination of the fractions of
doi:10.1074/jbc.272.3.1571
pmid:8999830
fatcat:obf77nibb5dtzkd4vvcg7tcw24