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.