The exchange of Fe3+ between acetohydroxamic acid and transferrin. Spectrophotometric evidence for a mixed ligand complex.
Transferrin, the serum iron transport protein, provides an excellent model for studying biological metal ion exchange reactions. A curious problem is that while a mixed ligand species of chelate-Fe3+-protein is anticipated from theoretical considerations and supported by kinetic results, no clear spectrophotometric evidence for such an intermediate has heretofore been obtained. In this study of the exchange of Fe3+ between acetohydroxamic acid and transferrin such evidence has been found. The reaction of Fe2+-acetohydroxamic acid with apotransferrin-CO3(2-) is distinctly biphasic when examined by stopped flow spectrophotometry. The first phase is complete within approximately 4 s and results in the formation of a transient species with a distinct spectral maximum at 432 nm. The second phase requires approximately 2 min and results in the formation of Fe3+-transferrin-CO3(2-). We suggest that the transient species is a mixed ligand complex. The reaction rate-concentration relationship for the formation of the intermediate is linear for Fe3+-acetohydroxamic acid and hyperbolic for apotransferrin-CO3(2-). This suggests a rate-limiting labilization of Fe3+-(acetohydroxamic acid)3 preceding attack by the apotransferrin-CO3(2-). The reverse reaction, the removal of Fe3+ from the Fe3+-transferrin-CO3(2-) by acetohydroxamic acid, does not provide spectral evidence for the intermediate. The velocity-concentration relationship shows a hyperbolic dependence on acetohydroxamic acid concentration and a linear dependence of Fe3+-transferrin-CO3(2-), suggesting a rate-limiting labilization of the Fe3+ of Fe3+-transferrin-CO3(2-) resulting from a conformational change.
D002254:Carbonates; D006801:Humans; D006877:Hydroxamic Acids; D007501:Iron; D007700:Kinetics; D011485:Protein Binding; D013053:Spectrophotometry; D014168:Transferrin / Q000378:metabolism*
D002254:Carbonates; D006877:Hydroxamic Acids; D014168:Transferrin; C006358:acetohydroxamic acid; D007501:Iron