A stopped-flow study of the cupric ion oxidation of adult-human haemoglobin

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Abstract
The addition of excess Cu2+ to adult human Hb leads to production of .alpha.22+.beta.23+, in the oxy and deoxy forms of the protein. Stopped-flow studies of the oxidation process yields apparent 2nd-order rate constants of 196 M-1.cntdot.s-1 and 41 M-1.cntdot.s-1 for the deoxy and oxy forms, respectively. The rate of the deoxy-form oxidation is linearly dependent on [Cu2+], whereas that of the oxy form is rate-limited above 2 mM to 0.11 s-1. Arrhenius activation energies of the 2 processes are almost identical at 91 kJ.cntdot.mol-1, as are the activation enthalpies of 89 kJ.cntdot.mol-1. The activation entropies show small differences, being 31 entropy units and 48 entropy units for the oxy and deoxy forms, respectively. ATP and glycerate 2,3-bisphosphate at saturating concentrations do not affect the rate of oxidation of the oxy form, but halve the rate found for the deoxy form. These data are discussed in terms of the previously proposed mechanism of oxidation in which slow Cu2+ binding is followed by rapid electron transfer.