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Ca2+-induced hydrophobic site on calmodulin: Application for purification of calmodulin by phenyl-Sepharose affinity chromatography

1982; Elsevier BV; Volume: 104; Issue: 2 Linguagem: Inglês

10.1016/0006-291x(82)90712-4

1090-2104

Rayudu Gopalakrishna, Wayne B. Anderson,

Enzyme Structure and Function

Calmodulin binds quantitatively to phenyl-Sepharose and octyl-Sepharose affinity columns in the presence of micromolar concentrations of Ca2+. In addition to EGTA, calmodulin also can be eluted from these affinity columns with low ionic strength buffer, non-ionic detergent (i.e., 1% Triton X-100), or ethylene glycol (50%), suggesting hydrophobic interaction. Using hydrophobic interaction chromatography calmodulin can be purified to homogeneity from bovine brain homogenate in a single step. For large-scale purification the protein fraction containing calmodulin was concentrated by isoelectric precipitation prior to application to the affinity column. The yield obtained by this procedure (160–180 mg calmodulin per kg brain) is significantly greater, and the time required (∼ 5 hr) is substantially less, than that of previously described procedures for calmodulin purification. It is apparent that phenyl-Sepharose offers several advantages over phenothiazine-Sepharose for affinity purification of calmodulin.