Alcohol-induced changes of {beta}-lactoglobulin - retinol-binding stoichiometry

It has been demonstrated using CD that ethanol induces importantsecondary structure changes of ß-lactoglobulin. CDspectra indicate that ß-lactoglobulin secondary structure,which is mainly composed of ß-strands, becomes mostly-helical under the influence of the solvent polarity changes.The midpoint of ß-strand/-helix transition in ß-lactoglobulinis observed at dielectric constant {small tilde}60 (35% ethanol;v/v). According to CD measurements, the ethanol-dependent secondarystructure changes are reversible. The alkylation of lysines-NH₂ in ß-lactoglobulin weakens the central ß-barrelstructure, since the ß-strand/-helix transition midpointof alkylated ß-lactoglobulin is shifted to lower ethanolconcentration (25% ethanol; v/v). ß-Lactoglobulinstructural changes are triggering the dissociation of the ß-lactoglobulin- retinol complex as judged from complete quenching of its fluorescencein ethanol concentration >30% (v/v). However, in 20% ethanol(v/v), ß-lactoglobulin still retains most of its nativesecondary structure as shown by CD and, in this condition, oneß-lactoglobulin molecule binds an additional secondretinol molecule. This suggests that the highly populated speciesobserved around 20% ethanol (v/v) might represent an intermediatestate able to bind two molecules of retinol.