Fluorescence Quenching As A Parameter For Measuring Complex Formation Between Metal Ions And Aromatic Amino Acids And Peptides

Abstract

The ultraviolet fluorescence of tryptophan and tyrosine is quenched when they are complexed with Cu⁺⁺ and Ni⁺⁺. Titrations of fixed amounts of the amino acids or their peptides with increments of metal ion performed in dilute buffered solutions, yield curves from which the binding constants of the 1:1 complexes can be determined by curve fitting. The association constants found by this method agree with those obtained by potentiometric titration. The binding of these metal ions to glycyl-L-tryptophan was also measured. Zn⁺⁺, while not quenching tryptophan fluorescence upon binding, competes with Cu⁺⁺, thus permitting the Zn⁺⁺-tryptophan association constant to be found. Unlike potentiometric titration, fluorescence quenching would detect binding even when the metal ion does not displace H⁺. Inherent limitations of the method involve collisional quenching and self absorption artifacts at high reactant concentrations.