Spectroscopic methods have shown that Ca2+ chelators interact with Ca2+-binding proteins. These spectral alterations have been interpreted as evidence for the binding of chelator by the proteins. We show by direct examination of EDTA interaction with calmodulin and α-lactalbumin that these proteins repel EDTA rather than bind it. The repulsion is reduced by increased salt concentration but is unaffected by Ca2+ binding to the proteins. The acidic protein, α-lactalbumin, repells the negatively charged EDTA and inorganic phosphate whereas the basic protein, lysozyme, repells the positively charged spermine. Thus, spectroscopic changes induced by negatively charged Ca2+ chelators on negatively charged Ca2+-binding proteins are due to electrostatic repulsion, and not to binding. These observations underscore the possible pitfalls of using spectroscopic methods alone to analyze protein-ligand interactions.
