There is some question whether the calcium binding characteristics of calciphorin are due to contaminating phospholipids. To differentiate protein ion binding by phospholipids or contaminating detergent, we describe here the use of Eu(III) as a metal-binding-site probe, and characterize the interaction of Eu(III) with calciphorin, cardiolipin, deoxycholate, and digitonin. The luminescence excitation pattern of Eu(III) bound to the calciphorin preparation clearly differentiates it from Eu(III) interactions with the possible contaminants. In addition, the effect of the luminescence decay constant of Eu(III) bound to calciphorin on the mole fraction of H2O in a mixture of H2O/2H2O indicates that all except approximately 0.8 of the 9 to 10 water molecules coordinating Eu(III) in solution are stripped off upon binding to calciphorin. This also contrasts with the data for the possible contaminants.
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