Distance measurements between the metal-binding sites in thermolysin using terbium ion as a fluorescent probe.

A single terbium ion has been introduced into thermolysin replacing two of the four calcium ions, and the fluorescence properties of the protein-bound terbium have been studied. The fluorescence of Tb⁺³ is tremendously enhanced (～7 × 10³) upon binding and is significantly quenched when divalent cobalt is substituted for the zinc ion normally found in the enzyme. By use of the Förster equation for energy transfer the distance between the protein-bound Tb⁺³ and Co⁺² in the active site was calculated to be 13.6±0.5 A. This agrees closely with the value of 13.9 A obtained from the crystal structure and suggests that energy transfer between the two metal ions bound to the protein takes place by a dipole-dipole mechanism.