TY - JOUR
T1 - Probing the hemoglobin central cavity by direct quantification of effector binding using fluorescence lifetime methods
AU - Gottfried, David S.
AU - Juszczak, Laura J.
AU - Fataliev, Nazim A.
AU - Seetharama Acharya, A.
AU - Hirsch, Rhoda Elison
AU - Friedman, Joel M.
PY - 1997
Y1 - 1997
N2 - 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 27 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 mol 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.
AB - 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 27 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 mol 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.
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U2 - 10.1074/jbc.272.3.1571
DO - 10.1074/jbc.272.3.1571
M3 - Article
C2 - 8999830
AN - SCOPUS:0031026292
SN - 0021-9258
VL - 272
SP - 1571
EP - 1578
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 3
ER -