T-STATE HEMOGLOBIN BINDS OXYGEN NONCOOPERATIVELY WITH ALLOSTERIC EFFECTS OF PROTONS, INOSITOL HEXAPHOSPHATE, AND CHLORIDE

Hemoglobin is the paradigm of allosteric proteins. Over the years, coo perative oxygen binding has been explained by different models predict ing that the T state of hemoglobin binds oxygen either noncooperativel y or with some degree of cooperativity or with strong cooperativity. T herefore, a critical test that discriminates among models is to determ ine the oxygen binding by the T state of hemoglobin. Fixation of hemog lobin 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 a ffinity compared with solution, with no Bohr effect and with no influe nce of other allosteric effecters. In this study, we have determined a ccurate oxygen-binding curves to the T state of hemoglobin in silica g els with the same microspectrophotometric apparatus and multiwavelengt hs 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 fi rst oxygen molecule. Other allosteric effecters such as inositol hexap hosphate, bezafibrate, and chloride significantly affect oxygen affini ty. Therefore, T state hemoglobins that are characterized by strikingl y different functional properties share the absence of cooperativity i n the binding of oxygen. These findings are fully consistent with the Monod, Wyman, and Changeux model and with most features of Perutz's st ereochemical model, but they are not consistent with models of both Ko shland and Ackers.