Fluorescence properties of terbium-alkaline phosphatase

The addition of Tb³⁺ to apoalkaline phosphatase at pH 8.0 results in the formation of a metalloprotein with an enhanced Tb³⁺ fluorescence at 492, 545, and 580 nm. The Tb³⁺ excitation spectrum is most consistent with a process in which energy is transferred from one or more tyrosyl chromophores to the bound lanthanide. An analysis of the fluorescence data under equilibrium conditions yields one Tb³⁺ binding site per enzyme dimer with a K_n = 0.16 ± 0.02 μm. The Tb³⁺-alkaline phosphatase complex is not catalytically active nor does it incorporate covalently bound phosphate, but the specific activity of Zn²⁺-alkaline phosphatase is significantly enhanced in the presence of Tb³⁺ indicating that this lanthanide mimics Mg²⁺ in stabilizing the structure of alkaline phosphatase. The fluorescence of the Tb³⁺-enzyme is found to be quite sensitive to conformational changes which occur upon addition of Zn²⁺ or substrates.