Probing the hemoglobin central cavity by direct quantification of effector binding using fluorescence lifetime methods.
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 approximately 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.
Gottfried
D S
DS
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA. gottfrie@aecom.yu.edu
Juszczak
L J
LJ
Fataliev
N A
NA
Acharya
A S
AS
Hirsch
R E
RE
Friedman
J M
JM
D000328:Adult; D001667:Binding, Competitive; D002263:Carboxyhemoglobin / Q000378:metabolism; D018360:Crystallography, X-Ray; D005456:Fluorescent Dyes; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D015335:Molecular Probes; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D006454:Hemoglobins; D015335:Molecular Probes; D002263:Carboxyhemoglobin