Stopped-flow kinetic studies of metal ion dissociation or exchange in a tryptophan-containing parvalbumin.
The rates of dissociation of 2 equiv of various metal ions [Ca(II), Cd(II), Pr(III), Nd(III), Sm(III), Eu(III), Gd(III), Tb(III), Dy(III), Ho(III), Er(III), Yb(III), and Lu(III)] from the primary CD and EF metal ion binding sites of parvalbumin (isotype pI = 4.75) from codfish (Gadus callarius L) were measured by stopped-flow techniques. The removal or replacement of metal ions was monitored by changes in sensitized Tb(III) luminescence or in intrinsic protein tryptophan fluorescence as quenching ions [Eu(III) or Yb(III)] were bound or removed or as the apoprotein was formed. In experiments wherein the bound metal ions were removed by mixing the parvalbumin with an excess of 1,2-diaminocyclohexanetetraacetic acid (DCTA), the kinetic traces were best fit by a double exponential with koff rate constants of 1.07 and 5.91 s-1 for Ca(II), 1.54 and 10.5 s-1 for Cd(II), and approximately 0.05 and approximately 0.5 s-1 for all of the trivalent lanthanide ions. In experiments wherein the bound metal ions were exchanged with an excess of a different metal ion, pseudo-first-order rate constants were proportional to the concentration of excess attacking metal ion for both the fast and slow processes in most experiments. In these cases, extrapolation of the rate constants to zero concentration of attacking metal ion gave values which agree well with the DCTA scavenging results. This finding demonstrates that the off rate constants do not depend on the occupancy of the neighboring site and therefore implies that there is no significant cooperativity in metal ion binding between the two sites in parvalbumin.
D000818:Animals; D002412:Cations; D005399:Fishes; D007700:Kinetics; D008433:Mathematics; D008670:Metals; D009124:Muscle Proteins / Q000378:metabolism*; D010320:Parvalbumins / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014364:Tryptophan
D002412:Cations; D008670:Metals; D009124:Muscle Proteins; D010320:Parvalbumins; D014364:Tryptophan