T State Hemoglobin Binds Oxygen Noncooperatively with Allosteric Effects of Protons, Inositol Hexaphosphate, and Chloride

Abstract

Hemoglobin is the paradigm of allosteric proteins. Over the years, cooperative oxygen binding has been explained by different models predicting that the T state of hemoglobin binds oxygen either noncooperatively or with some degree of cooperativity or with strong cooperativity. Therefore, a critical test that discriminates among models is to determine the oxygen binding by the T state of hemoglobin. Fixation of hemoglobin in the T state has been achieved either by crystallization from polyethylene glycol solutions or by encapsulation in wet porous silica gels. Hemoglobin crystals bind oxygen noncooperatively with reduced affinity compared with solution, with no Bohr effect and with no influence of other allosteric effectors. In this study, we have determined accurate oxygen-binding curves to the T state of hemoglobin in silica gels with the same microspectrophotometric apparatus and multiwavelengths analysis used in crystal experiments. The T state of hemoglobin in silica gels binds oxygen noncooperatively with an affinity and a Bohr effect similar to those observed in solution for the binding of the first oxygen molecule. Other allosteric effectors such as inositol hexaphosphate, bezafibrate, and chloride significantly affect oxygen affinity. Therefore, T state hemoglobins that are characterized by strikingly different functional properties share the absence of cooperativity in the binding of oxygen. These findings are fully consistent with the Monod, Wyman, and Changeux model and with most features of Perutz's stereochemical model, but they are not consistent with models of both Koshland and Ackers.Hemoglobin binds oxygen cooperatively with an affinity that is regulated by protons, organic phosphates, chloride, and carbon dioxide (1, 2). These properties are associated to the peculiar structure of the T quaternary state since the R quaternary state exhibits functional properties similar to those of isolated ␣-␤ dimers and isolated chains. For this reason, a great effort has been devoted to functionally and structurally characterize the T state and pathway from deoxyhemoglobin to oxyhemoglobin (1-4, 11, 12). Along this pathway, oxygen binding leads to tertiary conformational changes and to the quaternary transition whose relevance to the functional properties of hemoglobin is model-dependent. In fact, according to the Monod, Wyman, and Changeux model (5-7), hemoglobin exists in two quaternary states, a low affinity T state and a high affinity R state. Ligand binding to either the T or the R state is predicted to be noncooperative. The progressive shift of the T-R equilibrium in favor of the R state as a function of ligand saturation accounts for the positive cooperativity. The Monod, Wyman, and Changeux model has been extended to include the effect of allosteric ligands on the T state affinity (3, 8) and to fit nanosecond-millisecond kinetics of ligand binding as well as equilibrium data simultaneously (9). Furthermore, the extensive investigation of structural, spectroscopic, and functional properties of hemog...

Dynamics of Proteins Encapsulated in Silica Sol−Gel Glasses Studied with IR Vibrational Echo Spectroscopy

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“…The basic assumption underlying the use of BZF for hemoglobin research has been that the drug interacts only to T-state hemoglobin (13)(14)(15)(16). However, some evidence shows that this assumption is not correct.…”

UV Resonance Raman Spectra of Ligand Binding Intermediates of Sol-Gel Encapsulated Hemoglobin

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