Purification and peptide mapping of calmodulin and its chemically modified derivatives by reversed-phase...

Methods were developed for the isolation and peptide mapping of calmodulin and its chemically modified derivatives by reversed-phase high-performance liquid chromatography (HPLC). Calmodulin and its guanidinated, iodinated, and performic acid-oxidized derivatives can be isolated on alkylphenyl columns by using gradients of acetonitrile in 10 mM potassium phosphate, pH 6.0, 2 mM EGTA. Peptide mapping by HPLC, following complete digestion of the proteins with clostripain, allows identification of the modified amino acids residues. Clostripain peptides are eluted in the order 87-90, 75-86, 91-106, 107-126, 127-148, 107-148, 1-37, and 38-74. Performic acid oxidation of methionines decreases the retention times of the modified peptides, whereas iodination of tyrosines or guanidination of lysines increases retention times of modified peptides. These HPLC methods are applicable to the identification of specific modifications of calmodulin, allowing the assessment of the role of individual amino acid residues in determining the unique physical, chemical, and spectroscopic properties of this ubiquitous intracellular calcium-binding protein.