Fluorescence energy-transfer measurements between the calcium binding site and the specificity pocket of bovine trypsin using lanthanide probes

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Abstract
Using fluorescence energy-transfer experiments the distance between the specificity pocket and the Ca2+ binding site of bovine pancreatic trypsin was measured. Proflavin and thionine were used to block the specificity site, whereas various lanthanide ions were substituted for the Ca. It was then possible to choose various donor-acceptor pairs which exhibited suitable energy transfer. The distance between proflavin and Nd(III), Pr(III) and Ho(III) were calculated to be 10.9, 10.3 and 10.3 .ANG., respectively. This agrees very well with the value of approximately 10 .ANG. obtained between the methyl protons of p-toluamidine (a competitive inhibitor) and Gd(III) using NMR techniques. This is strong evidence that in solution the Ca binding site is composed of the side chains of Ser-190 and Asp-194.