The properties of human methemoglobin have been investigated under a wide variety of conditions to determine its conformation and to test for evidence of the T state conformation which has been proposed by Perutz to exist in the presence of high spin ligands and inositol hexaphosphate (IHP). Subunit dissociation was measured as a criterion for the T state since marked differences in the tetramer-dimer equilibrium exist for oxyhemoglobin (R state) and deoxyhemoglobin (T state). In the absence of IHP, complexes of methemoglobin with both high spin ligands (water, fluoride) or low spin ligands (azide, cyanide) show extensive dissociation in 2,2-bis(hydroxymethyl)-2,2',2"-nitriloethanol buffers, pH 6, 0.1 M NaCl, with values of the tetramer-dimer dissociation constant (K4,2) near 10-5 M. The addition of IHP lowers K4,2 to a value near 10-5 M for all forms of methemoglobin. Combination of IHP with methemoglobin promotes a conformational change, but the change is apparently independence of spin state. The conformation acquired in the presence of IHP is not identical with the T state (K4,2 similar to 10-12 M) and can also occur with hemoglobin in the ferrous form, as revealed by a substantial reduction in K4,2 for CO-hemoglobin upon addition of IHP. Subunit dissociation has also been measured using the haptoglobin reaction, since haptoglobin binds only to hemoglobin dimers. The haptoglobin experiments give results that are qualitatively in agreement with the conclusions reached by ultracentrifuge measurements. Similar results are also obtained by estimating the degree of dissociation on the basis of the material which aggregates following mixing with dithionite. The effect of IHP on azide-binding kinetics with methemoglobin has also been examined. Changes in reactivity is observed upon addition of IHP, but the principal effect is observed upon addition of IHP, but the principal effect is an enhancement of the rate of reaction of the beta chains. Changes in the reactivity of the beta93 sulfhydryl group of methemoglobin also accompany addition of IHP, but in a manner which is largely independent of the spin state of the iron. Similar changes are again found with CO-hemoglobin upon addition of IHP. The rate of binding of bromthymol blue also shows some changes upon addition of IHP, but the changes are more pronounced for deoxyhemoglobin than for methemoglobin. Since the results obtained did not appear to indicate a significant role for spin state in the changes observed, additional studies were undertaken using EPR spectroscopy.

Azides↗

PubMed1975

Magnitude of subunit inequivalence for oxygen release from hemoglobin: reinvestigation of the oxygen-pulse experiment.

Two hypotheses have been presented to explain the grossly biphasic oxygen release kinetics observed when hemoglobins are studied with the oxygen pulse technique [Gibson (1973) Proc. Nat. Acad. Sci. USA 70, 1-4]. Hypothesis I suggests that the two phases result from cooperativity, with the fast phase being oxygen release from the low affinity (T) state and the slow phase being oxygen release from molecules that have switched to the high affinity (R) state. Hypothesis II suggests that the biphasic curves are due to a large (factor of 20-30) difference in oxygen release from the two types of subunits within deoxyhemoglobin. In order to experimentally discriminate between these two hypotheses, we reinvestigated the oxygen pulse reaction for hemoglobin Kansas (alpha2 beta2 102 Asn leads to Thr) in the absence and presence of inositol hexaphosphate, since recent high resolution nuclear magnetic resonance studies have shown that this allosteric cofactor stabilizes hemoglobin Kansas in T even when fully liganded [Ogawa, Mayer, and Shulman (1972) Biochem. Biophys. Res. Commun. 49, 1485-1491]. The results of these studies clearly favor hypothesis I over hypothesis II as being the correct interpretation for the oxygen pulse results. However, we have found evidence that suggests that oxygen release and binding in T are surprisingly faster than previously observed. Furthermore, within T, there is some spectral and kinetic heterogeneity for oxygen release from adult hemoglobin and hemoglobin Kansas. The magnitude of this kinetic heterogeneity in T appears to be about the same as that seen in the high affinity, R, state. The exchange of hypothesis II for hypothesis I more strongly favors views of cooperative oxygen binding involving both types of subunits, as required if the allosteric model of Monod, Wyman, and Changeux [(1965) J. Mol. Biol. 12, 88-118] is considered operative.

Adult↗

PubMed1974