Separation of Tyrosine Phosphorylated Calmodulin from Calmodulin Using Non-Porous Anion-Exchange HPLC

Abstract

Calmodulin is a protein which exerts control over various physiological processes by binding and modulating key enzymes after it undergoes a calcium-induced conformational change. The insulin receptor kinase has been shown to phosphorylate tyrosine 99, in the third calcium binding cleft of the protein. Since each molecule accommodates only four calcium ions, the introduction of phosphate into one of these calcium binding pockets may alter biological activity. To determine the effects of this modification, required the separation of phosphorylated calmodulin from the unphosphorylated form.

This paper addresses the HPLC techniques we have used for the successful separation of various forms of calmodulin. Reversed-phase HPLC at pH 7.8 in the presence of 100μM CaCl₂ or 1mM EGTA gave different elution profiles, reflecting the effect Ca⁺⁺ has on calmodulin conformation. Such changes were not observed using 0.1% trifluoroacetic acid in mobile phases since calmodulin binds essentially no calcium at pH 2.0. Anion-exchange HPLC on a non-porous DEAE-type resin was found to be superior to reversed-phase methods for the specific separation of phosphorylated calmodulin from unphosphorylated calmodulin.