Nonequivalent Binding to Human Methemoglobin

• Published in: The Journal of biological chemistry Vol. 246; no. 22; pp. 6849 - 6854
• Main Authors: Klapper, Michael H., Uchida, Hiroshi
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 25.11.1971
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)45924-3

The reactions of thiocyanate and fluoride with the iron of methemoglobin A may each be analyzed with a single association constant at pH 6.0 and at 22°. This implies that the four protein sites bind independently and with identical intrinsic affinities. In contrast the reactions of cyanide and imidazole under the same experimental conditions require two association constants for a minimal description of their binding isotherms. This apparent contradiction may be resolved by making the formal assumption that the high-low spin equilibria of the protein irons are not identical. The reaction kinetics of these four iron-coordinating ligands display a different pattern. Fluoride and cyanide react with a simple, pseudo-first order time dependence. Thiocyanate and imidazole show biphasic kinetics, requiring two constants to adequately describe the results. This lack of correspondence between kinetic and thermodynamic results suggests the ligand-binding mechanism contains a minimum of two reaction steps. This conclusion is verified by the lack of agreement between thermodynamically and kinetically calculated association constants, and the observation of a mixed order dependence of the thiocyanate reaction on ligand concentration.