Rates for the R leads to T conformational change of deoxyhemoglobin formed by laser photolysis of carboxyhemoglobin were determined from CO rebinding observed in three solution systems with viscosities between 1 and 6 cP. Experiments were carried out at 20 degrees C and pH 8.3 in solutions consisting of borate buffer containing various amounts of sucrose, glycerol, or ethylene glycol. As in the case of earlier experiments in borate buffer (Sawicki and Gibson, 1976, J. Biol. Chem., 251:1533-1542), a simple two-state allosteric model which takes into account tetramer-dimer dissociation was found to give a good description of all experimental results. Using measured values for the R- and T-state CO-binding rate constants and the tetramer-dimer dissociation constant, values for the conformational change rate were determined by fitting this model to the experimental data. These rates were compared with Gavish's transient strain model (Gavish, 1978, Biophys. Struct. Mech., 4:37-52), which predicts an inverse dependence of conformational change rate on viscosity. Although fair agreement is found for hemoglobin in sucrose/borate solutions, in glycerol/borate and ethylene glycol/borate solutions, conformational change rate falls off much more rapidly with increasing viscosity than predicted by the model.
The generally accepted value for the tetramer-dimer dissociation constant KL4,2 of carboxyhemoglobin in pH 7.0 phosphate buffer lies in the range 1--2 micrometers. Previous determinations of the quantity have generally involved addition of dithionite to samples to exclude oxygen. We report flash photolysis experiments on carboxyhemoglobin in the absence of dithionite which suggest that KL4,2 is 0.2 +/- 0.05 micrometer. Addition of dithionite to our samples resulted in an order of magnitude increase in KL4,2 in good agreement with previously published results. The mechanism of this increase in dissociation has not been determined with certainty. However, impurities, possibly metal ions, are required in addition to dithionite to produce this effect. Dithionite did not increase KL4,2 for phosphate buffer solutions treated with Chelex 100 analytical grade chelating resin. Addition of bovine serum albumin to untreated buffer solutions before addition of dithionite was found to prevent increased dissociation. The sulfhydryl-reducing agents dithiothreitol and beta-mercaptoethanol were found to protect against the effect of dithionite and to reverse its effect on KL4,2 if they were added after the dithionite. The interaction of the unknown impurities with dithionite to produce increased values of KL4,2 could be mimicked by addition of CU2+ ions in concentrations of less than 1 micrometer to buffer treated with Chelex 100 resin.
The spectral difference between normal and rapidly reacting deoxyhemoglobin (Sawicki and Gibson (1976), J. Biol Chem. 251:1533-1542) is used to study the relationship between CO binding to hemoglobin and the conformational changes to the rapidly reacting form in a combined flow-laser flash experiment. In both pH 7 phosphate buffer and pH 7 bis(2-hydroxy-ethyl)imino-tris (hydroxymethyl)methane buffer (bis-Tris) with 500 muM 2,3-diphosphoglycerate (DPG), the conformational change lags far behind CO binding; rapidly reacting hemoglobin is not observed until more than 10% of the hemoglobin is liganded. In pH 9 borate buffer the formation of rapidly reacting hemoglobin leads CO binding by a significant amount. A simple two-state allosteric model (Monod et. al. (1965), J. Mol. Biol. 12:88-118) which assumed equivalence of the hemoglobin subunits in their reaction with CO was used to simulate the experimental results. In terms of the model, the conformational change lead observed at pH 9 suggests that significant conformational change has occurred after binding of only one CO molecule per tetramer. In the presence of phosphates good agreement between experimental results and simulations is obtained using parameter values suggested by previous experimental studies. The simulations suggest that the conformational change occurs after binding of three CO molecules.
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