Melittin-silica, a high-pressure affinity chromatography resin for calmodulin

Calmodulin binds the peptide melittin with nanomolar affinity in the presence of Ca2+ but not in its absence. Melittin was coupled to glycidyloxypropyl-silica and used for the high-performance affinity chromatography of calmodulin. Calmodulin was retained by the resin in Ca2+-containing buffers and eluted in Ca2+-free buffers. Columns prepared from resin coupled to melittin in a batch procedure gave superior performance to those prepared under flow. Column capacity was 1.6 mg calmodulin/g resin for resins containing 1.4 mg melittin/g. Melittin-silica selectivity bound calmodulin and did not bind similar proteins such as troponin C or parvalbumin, however, other results suggest that at least one non-calmodulin protein in brain may also bind to the resin. Chromatography of crude pea plant extracts yielded homogeneous calmodulin. By modifying the standard elution protocol and/or using post-column reaction with o-phthalaldehyde, detection of less than 50 ng calmodulin in 7 min total analysis time was demonstrated. The column was used to investigate hormone- and light-induced changes in the calmodulin levels in pea plants.