Spectroscopic Analysis of the Cu2+-Induced Fluorescence Quenching of Fluorescent Proteins AmCyan and mOrange2

Abstract

Fluorescent proteins show fluorescence quenching by specific metal ions, which can be applied towards metal biosensing applications. In order to develop metal-biosensor, we performed spectroscopic analysis of the fluorescence quenching of fluorescent protein AmCyan and mOrange2 by various metal ions. The fluorescence intensity of AmCyan was reduced to 48.54% by Co2+ and 67.77% by Zn2+; Cu2+ reduced the fluorescence emission of AmCyan to 19.30% of its maximum. The fluorescence intensity of mOrange2 was quenched by only Cu2+, to 11.48% of its maximum. When analyzed by Langmuir equation, dissociation constants for AmCyan and mOrange2 were 56.10 and 21.46 A mu M, respectively. The Cu2+ quenching of AmCyan and mOrange2 were reversible upon treatment with the metal chelator EDTA, indicating that the metal ions were located on the protein surface. Their model structures suggest that AmCyan and mOrange2 have novel metal-binding sites.