Lanthanide ions induce hydrolysis of hemoglobin-bound 2,3-diphosphoglycerate (2,3-DPG), conformational changes of globin and bidirectional changes of2,3-DPG-hemoglobin's oxygen affinity

The changes in structure and function of 2,3-diphosphoglycerate-hemoglobin (2,3-DPG-Hb) induced by Ln(3+) binding were studied by spectroscopic method s. The binding of lanthanide cations to 2,3-DPG is prior to that to Hb. Ln( 3+) binding causes the hydrolysis of either one from the two phosphomonoest er bonds in 2,3-DPG non-specifically. The results using the ultrafiltration method indicate that Ln(3+) binding sites for Hb can be classified into th ree categories: i.e. positive cooperative sites (N-I), non-cooperative stro ng sites (N-S) and non-cooperative weak sites (N-W) with binding constants in decreasing order: K-I > K-S > K-W. The total number of binding sites amo unts to about 65 per Hb tetramer. Information on reaction kinetics was obta ined from the change of intrinsic fluorescence in Hb monitored by stopped-f low fluorometry. Fluctuation of fluorescence dependent on Ln(3+) concentrat ion and temperature was observed and can be attributed to the successive co nformational changes induced by Ln(3+) binding. The results also reveal the bidirectional changes of the oxygen affinity of Hb in the dependence on Ln (3+) concentration. At the range of [Ln(3+)]/[Hb] < 2, the marked increase of oxygen affinity (P-50 decrease) with the Ln(3+) concentration can be att ributed to the hydrolysis of 2,3-DPG, while the slight rebound of oxygen af finity in higher Ln(3+) concentration can be interpreted by the transition to the T-state of the Hb tetramer induced by Ln(3+) binding. This was indic ated by the changes in secondary structure characterized by the decrease of <alpha>-helix content. (C) 2001 Elsevier Science B.V. All rights reserved.