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J. Biol. Chem., Vol. 281, Issue 36, 25972-25983, September 8, 2006

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Allosteric Effectors Influence the Tetramer Stability of Both R- and T-states of Hemoglobin A^*

Gusztáv Schay, László Smeller, Antonio Tsuneshige, Takashi Yonetani, and Judit Fidy¹

From the Department of Biophysics and Radiation Biology and Biophysics Research Group of the Hungarian Academy of Sciences, Faculty of Medicine, Semmelweis University, P. O. Box 263 H 1444 Budapest, Hungary and the Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine and the Johnson Research Foundation, Philadelphia, Pennsylvania 19104-6059

The contribution of heterotropic effectors to hemoglobin allostery is still not completely understood. With the recently proposed global allostery model, this question acquires crucial significance, because it relates tertiary conformational changes to effector binding in both the R- and T-states. In this context, an important question is how far the induced conformational changes propagate from the binding site(s) of the allosteric effectors. We present a study in which we monitored the interdimeric interface when the effectors such as Cl^–, 2,3-diphosphoglycerate, inositol hexaphosphate, and bezafibrate were bound. We studied oxy-Hb and a hybrid form (FeO₂)₂-(Zn)₂ as the T-state analogue by monitoring heme absorption and Trp intrinsic fluorescence under hydrostatic pressure. We observed a pressure-dependent change in the intrinsic fluorescence, which we attribute to a pressure-induced tetramer to dimer transition with characteristic pressures in the 70–200-megapascal range. The transition is sensitive to the binding of allosteric effectors. We fitted the data with a simple model for the tetramer-dimer transition and determined the dissociation constants at atmospheric pressure. In the R-state, we observed a stabilizing effect by the allosteric effectors, although in the T-analogue a stronger destabilizing effect was seen. The order of efficiency was the same in both states, but with the opposite trend as inositol hexaphosphate > 2,3-diphosphoglycerate > Cl^–. We detected intrinsic fluorescence from bound bezafibrate that introduced uncertainty in the comparison with other effectors. The results support the global allostery model by showing that conformational changes propagate from the effector binding site to the interdimeric interfaces in both quaternary states.

Received for publication, May 2, 2006 , and in revised form, June 23, 2006.

^* This work was supported by a collaborative grant from the Fogarty International Center, Award TW005924 (to T. Y. and J. F.), National Science Foundation of Hungary Grants OTKA T049213 (to L. S.) and OTKA TS-044730 (to J. F.), by NHLBI Grant HL14508 from the National Institutes of Health (to T. Y.), and by a Ph.D. fellowship from Eötvös University, Budapest (to J. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Fax: 36-1-2666656; E-mail: judit{at}puskin.sote.hu.

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