Binding of Lanthanides and of Divalent Metal Ions to Porcine Trypsin

The single binding site for Ca²⁺ on the porcine trypsin molecule was found to be capable of binding trivalent lanthanide ions as well as Y3+ and the divalent ions Mn²⁺ and Cd²⁺. The affinity of the lanthanide ions to trypsin depends strongly on their ionic radius; the smaller the lanthanide ion, the higher the binding constant. The affinity of metal ions to trypsin does not depend on their chemical similarity to Ca²⁺. The fluorescence of Tb³⁺ is enhanced 10⁵-fold when bound to porcine trypsin. The pH dependence of the Tb³⁺ fluorescence indicates that the metal binding site of the enzyme consists of one or more carboxyl side chains. Measurements on the fluorescence excitation spectrum of the trypsin-Tb³⁺ complex reveal energy transfer from one or more tryptophans in close proximity to the bound metal ion.