High performance capillary electrophoresis of calmodulin

The electrophoretic properties of purified calmodulin were investigated. High performance capillary electrophoresis of this Ca2+(-)binding protein in free solution at pH 2.5 resulted in an elution of a single peak with a retention time of approximately 4.7 min. Addition of ethylene-bis(oxyethylenenitrilo]) N,N,N',N'-tetraacetic acid (EGTA) to the protein prior to capillary electrophoresis completely abolished this electrophoretic profile. Polyacrylamide gel electrophoresis of calmodulin under denaturing and nondenaturing conditions also revealed a single polypeptide band. However, the relative electrophoretic mobilities of this protein could vary, depending on the presence or absence of Ca2+. The pI of calmodulin was estimated to be 3.7 by using isoelectric focusing techniques. Analysis of this acidic protein by high performance capillary electrophoresis at pH 8.0 revealed that it could be resolved into two major and one minor polypeptide peaks, regardless of the presence or absence of Ca2+. These findings suggest that capillary electrophoresis at near physiological pH may differentiate the microheterogeneity of calmodulin.