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Abstract
The formation of deoxyhemoglobin was examined by measuring the heme spectral change that accompanies the aggregation of isolated alpha and beta chains. At low hemeconcentrations (less than 10(-5) M), tetramer formation can be described by two consecutive, second order reactions representing the aggregation of monomers followed by the association of alphabeta dimers. At neutral pH, the rates of monomer and dimer aggregation are roughly the same, approximately 5 X 10(5) M(-1) X(-1) at 20 degrees. Raising or lowering the pH results in a uniform decrease of both aggregation rates due presumably to repulsion of positively charged subunits at acid pH and repulsion of negatively charged subunits at alkaline pH. Addition of p-hydroxymercuribenzoate to alpha chains lowers the rate of monomer aggregation whereas addition of mercurials to the beta subunits appears to lower both the rate of monomer and the rate of dimer aggregation. At high heme concentrations (greater than 10(-5) M) or in the presence of organic phosphates, the rate of chain aggregation becomes limited, in part, by the slow dissociation of beta chain tetramers. In the case of inositol hexaphosphate, the rate of hemoglobin formation exhibits a bell-shaped dependence on phosphate concentration. When intermediate concentrations of inositol hexaphosphate (approximately 10(-4 M) are preincubated with beta subunits, a slow first order time course is observed and exhibits a half-time of about 8 min. As more inositol hexaphosphate is added, the chain aggregation reaction begins to occur more rapidly. Eventually at about 10(-2) M inositol hexaphospate, the time course becomes almost identical to that observed in the absence of phosphates. The increase in the velocity of the chain aggregation reaction at high phosphate concentrations suggests strongly that inositol hexaphosphate binds to beta monomers and, if added in sufficiently large amounts, promotes beta4 dissociation. A quantitative analysis of these results showed that the affinity of beta monomers for inositol hexaphosphate is the same as that of alphabeta dimers. Only when tetramers are formed, either alpha2beta2 or beta4, is a marked increase in affinity for inositol hexaphosphate observed.
Highlights
The increase in the velocity of the chain aggregation reaction at high phosphate concentrations suggests strongly that inositol hexaphosphate binds to p monomers and, if added in sufficiently large amounts, promotes p4 dissociation
The total absorbance change was directly proportional to the a-heme concentration when the p subunits were present in excess (Fig. 1A) and to P-heme concentration when the (Y subunits were in excess (Fig. 1B)
These results show that both chain preparations are fully reactive with respect to hemoglobin formation and that there are no significant deviations from Beer’s law over the heme concentration range used (2 to 15 x 10m6 M)
Summary
At high heme concentrations (>lO-” M) or in the presence of organic phosphates, the rate of chain aggregation becomes limited, in part, by the slow dissociation of /3 chain tetramers. As more inositol hexaphosphate is added, the chain aggregation reaction begins to occur more rapidly. The increase in the velocity of the chain aggregation reaction at high phosphate concentrations suggests strongly that inositol hexaphosphate binds to p monomers and, if added in sufficiently large amounts, promotes p4 dissociation. ’ Abbreviations used are: Hb*, a rapidly reacting high affinity conformation of hemoglobin (an R state in terms of the allosteric, two-state model) whichhas been assigned to isolated chains, dimers, the last heme site to react with ligands (Hb,X,), and certain high affinity mutant and chemically modified hemoglobins [6]; inositol-
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