Calcium Binding Site of Trypsin as Probed by Lanthanides

A number of physicochemical techniques have been applied to identify, locate, and characterize the Ca²⁺-binding site in porcine and bovine trypsin. The fluorescent lanthanide Tb³⁺ and the paramagnetic lanthanide Gd³⁺ were used as probes for the Ca²⁺-binding site in the trypsin molecule. The fluorescent lanthanide Tb³⁺ was found to bind to the specific Ca²⁺-binding site on the trypsin molecule concomitant with a large increase in its fluorescence. The pH dependence of the Tb³⁺-binding process to the trypsin molecule and studies on proton release upon Tb³⁺ binding to the protein reveal the involvement of two carboxyl residues in metal binding. Proton relaxation rate measurements on the Gd³⁺-trypsin complex reveal that upon metal binding six of the eight water molecules coordinating the aquo-Gd³⁺ ion are released where two water molecules remain bound to the protein-bound Gd³⁺ ion. Model building of the trypsin molecule identifies the two carboxylate residues at the metal-binding site as Glu-70 and Glu-80, as recently revealed by x-ray crystallographic studies. Fluorescence excitation studies on the trypsin-Tb³⁺ complex reveal energy transfer from a tryptophan residue to the bound Tb³⁺ ion. This residue is identified as Trp-141. Trypsin was also found to possess a low-affinity site for lanthanide ions which is incapable of binding Ca²⁺. The existence of a secondary lanthanide-binding site is responsible for the variation of the circular polarized luminescence spectrum of the Tb³⁺-trypsin complex with the Tb³⁺ to protein ratio. Some differences are found between the spectroscopic properties of the lanthanide complexes of bovine trypsin and porcine trypsin. These differences stem from the structural differences of the Ca²⁺-binding sites of the two types of trypsins.