Development of a method to assess binding of astaxanthin to Atlantic salmon Salmo salar L. muscle proteins

Abstract

Several methods were examined to characterize the binding between astaxanthin and salmon muscle protein(s) in order to provide tools for evaluation of the role of muscle proteins on astaxanthin retention in Atlantic salmon Salmo salar L. £esh. The methods included gel ¢ltration chromatography, displacement of a hydrophobic probe and ultra¢ltration. With gel ¢ltration chromatography, aggregation of astaxanthin under the experimental conditions was a major problem for the separation of bound astaxanthin from free astaxanthin because the apparent molecular weight of aggregated astaxanthin or astaxanthin micelles was in the range of protein^astaxanthin complexes. Displacement of the £uorescent probe 8-anilino-1-naphthalenesulphonate (ANS) was not eiective as astaxanthin quenched the £uorophore so that displacement could not be observed. An ultra¢ltration method was developed using 200-mM sodium cholate for dispersion of astaxanthin aggregates. This allowed unbound astaxanthin to be separated from bound astaxanthin using a 30-kDa ¢lter. After salmon muscle proteins were solubilized in different fractions by sequential extraction using low ionic strength solutions, the astaxanthin binding of diierent fractions was assessed using the ultra¢ltration method. The signi¢cant diierence (Po0.05) observed in the astaxanthin binding of the various fractions suggests an application of this assay to detect diierences in a⁄nity of proteins for astaxanthin. The results also suggest that proteins other than actomyosin or actin can bind astaxanthin in Atlantic salmon £esh. This method can be used for the identi¢cation of astaxanthin-binding proteins in salmon £esh and other tissues.