Spectrophotometric evidence for a polar interaction betweenα-tocopherol and phospholipids: the effects of different phosphatides and mineral salts

Abstract

Previous investigations of a-tocopherol-phospholipid interactions have emphasized the hydrophobic effect and the steric fit between the phytyl sidechain of a-tocopherol and a polyunsaturated acyl sidechain in the phospholipid (Diplock & Lucy, 1973). The spectral properties of a-tocopherol in aqueous dispersions with phospholipids suggest, however, that a polar interaction also occurs involving the phenolic headgroup of the tocopherol and presumably the polar headgroup of the phospholipid. Thus, the absorbance maximum of a-tocopherol showed a hypsochromic shift to 291.5nm ( E = 3220) in an aqueous dispersion with soya bean lecithin compared with 297 nm ( E = 3870) in cyclohexane. The fluorescence intensity of a-tocopherol in cyclohexane (40pg of a-tocopherol/ml) was 2.0 arbitary units (Amax. emission 328nm). This was quenched to 0.50 (Amax. emission 328nm) in the aqueous lecithin dispersion (40pg of a-tocopherol/ml; 80pg of lecithin/ml). Similar quenching was observed in dispersions with several purified phospholipids including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol. In each case, the wavelengths of maximum excitation and emission were similar to those in the soya bean lecithin. Addition of mineral salt solutions to the a-tocopherol-phospholipid dispersions led to a progressive recovery of the a-tocopherol fluorescence. Fig. 1 shows data obtained with soya bean lecithin and KCl. A plot of the increased fluorescence (AF) against KCI concentration (0-200 mM) approximated to a hyperbola and the double-reciprocal plot was a straight line (Fig. l a ) with a positive intercept of they axis at and a negative intercept of the x axis corresponding to l /Kf, the apparent binding constant for KCI. When these experiments were repeated using different quantities of a-tocopherol, a series of double-reciprocal plots were obtained which made the same intercept on t h e y axis and gave a series of apparent binding constants for KCI ranging from K, = 8 3 m ~ with 20pg of a-tocopherol/ml to K, = 2 2 m ~ with 200pg of a-tocopherol/ml (Fig. la). Alternatively, when the a-tocopherol concentration was kept constant and the lecithin concentration was changed, a series of double-reciprocal plots were obtained which made the same intercept on the x axis but gave a series of intercepts on the y axis (Fig. lb). These and further experiments showed that the Kf for KCI in this experimental system was controlled by the a-tocopherol concentration and it was independent of the lecithin concentration. The value of AF,,,., on the other hand, was controlled by the lecithin concentration, but it was independent of the a-tocopherol concentration. Similar experiments were carried out using various purified phospholipids, as well as dicetyl phosphate which lacks the alcohol moiety of the phospholipid molecule. They each formed