Metal-ion-dependent hydrophobic-interaction chromatography of alpha-lactalbumins

Abstract

alpha-Lactalbumins from bovine, human, goat, sheep, and horse milk bind to phenyl-Sepharose in the presence of EDTA and can be eluted by addition of Ca2+ (0.001-100 mM). This property has been utilized to purify these proteins in a one-step purification from milk whey. alpha-Lactalbumin purified in this manner has the same ultraviolet and proton nuclear magnetic resonance spectra as that purified by other methods. Using binding to phenyl-Sepharose as an assay, the conformation of bovine alpha-lactalbumin upon the addition of several metal ions that are known to interact with this protein was investigated. Lanthanides, Mn2+, Mg2+, and Cd2+ can substitute for Ca2+, whereas Zn2+, Al3+, and Co2+ cannot. Surprisingly, whereas lower concentrations of La3+, Mn2+, and Cd2+ (1 mM and less) caused elution from the hydrophobic support, higher concentrations (10 mM) were ineffective. These observations can be rationalized assuming the presence of two distinct metal-ion binding sites with different specificities.