Calcium-dependent hydrophobic chromatography of calmodulin, S-100 protein and troponin-C

We have demonstrated calcium-dependent hydrophobic interactions among calmodulin, S-100 protein and troponin-C and a homologous series of ω-aminoalkyl-agaroses. The three Ca 2+ -binding proteins were retained on the column of agarose substituted with ω-aminooctyl or even longer with alkylamine, in the presence of Ca 2+ and 0.15 M NaCl. As these proteins were not retained on the column with shorter alkylamine ‘arms’ ( N = 2, 4), they are probably successively absorbed with a higher affinity to the hydrophobic agarose column. Calmodulin and S-100 protein were eluted from the aminooctyl-agarose column with 1 mM EGTA in the presence of 0.15 M NaCl and the elution of troponin-C was Ca 2+ -independently carried out with 0.3 M NaCl. On the other hand, S-100 and troponin-C were eluted Ca 2+ -dependently from aminodecyl-agarose in the presence of 1 M NaCl and half the amount of the calmodulin applied was eluted with 1 M NaCl. As there are obvious differences among the three Ca 2+ -binding proteins with regard to chromatographic behavior on ω-aminoalkyl-agarose columns, our results suggest that these three proteins expose different hydrophobic regions following Ca 2+ -induced conformational changes and, if so, such would explain the interaction with aminoalkyl-agaroses.