Stabilization of the T-state of ferrous human adult and fetal hemoglobin by Ln(III) complexes: a thermodynamic study.

The effect of the lanthanide(III) complexes [Gd(1,4,7,10-tetraazacyclododecane-N,N',N", N"'-tetrakis(methylenephosphonate))]5- (Gd-DOTP) and La-DOTP on the oxygen binding and spectroscopic properties of human adult and fetal hemoglobin (HbA and HbF, respectively) has been investigated. The affinity of Gd-DOTP and La-DOTP for oxygenated HbA (HbAO2; KHbAO2 = 2.6 x 10(-3) M) is closely similar to that observed for Ln(III) complexes association to nitrosylated HbA (HbANO KHbANO = 1.8 x 10(-3) M) and to aquo-met HbA (met-HbA; Kmet-HbA = 1.9 x 10(-3) M), being lower than that determined for Gd-DOTP and La-DOTP binding to the deoxygenated form of the tetramer (HbAd; KHbAd = 3.0 x 10(-4) M). The affinity of Gd-DOTP for deoxygenated HbF (HbFd; KHbFd = 9.5 x 10(-4) M) and oxygenated HbF (HbFO2; KHbFO2 = 3.7 x 10(-3) M) is lower than that observed for Ln(III) complexes association to HbAd and HbAO2, respectively. Gd-DOTP and La-DOTP bind to HbA and HbF with a 1:1 stoichiometry per tetramer. Increasing Gd-DOTP and La-DOTP concentration, oxygen affinity for HbA decreases (i.e. P50 increases), this effect being minor for HbF. Upon binding of Ln(III) complexes to HbANO, the X-band EPR spectrum and the absorption spectrum in the Soret region display the characteristics which have been attributed to the T-state of the ligated tetramer. These results represent a clear cut evidence for the specific binding of Gd-DOTP and La-DOTP to the 2,3-D-glycerate bisphosphate (BPG) pocket (i.e. at the dyad axis, in between the beta-chains) of HbA and HbF. The effect of Ln(III) complexes on the ligand binding and spectroscopic properties of HbA and HbF is reminiscent that of BPG, the physiological modulator of human Hb action.