Measurement of Interpeptidic Cu(II) Exchange Rate Constants by Static Fluorescence Quenching of Tryptophan.

Abstract

The interpeptidic exchange of Cu(II) between biologically relevant peptides like Gly-His-Lys (GHK) was measured through proximity static fluorescence quenching of a noncoordinating tryptophan residue by Cu(II). The inability to spectrally distinguish between starting and final Cu(H-1GHK)+ complexes by the current methods was solved by the replacement of noncoordinating lysine for tryptophan in the starting complex, Cu(H-1GHW). Because the apoGHW is the only fluorescent species, the recovered fluorescence is directly proportional to the [Cu(II)]exchanged between GHW and GHK. The apparent second-order rate constants of the exchanges from Cu(H-1GHW) to GHK and DAHK are 1.6 (±0.2) × 102 and 5.0 (±0.7) × 101 M-1 s-1, respectively. The easy-to-implement kinetic fluorescent method described here for Cu(II) interpeptidic exchange can be expanded to other biological systems.