Interaction of curcumin with phosphatidylcholine: A spectrofluorometric study.

Curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3, 5-dione], the main constituent of the rhizomes of the plant Curcume longa L. (turmeric), is a powerful antioxidant in both enzymatic and nonenzymatic systems. The interactions of curcumin with egg and soy phosphatidylcholine were followed by fluorescence spectroscopy. Curcumin had very weak fluorescence in aqueous system, which was enhanced in apolar environments. Curcumin emitted at 490 nm after being excited at 451 nm in phosphatidylcholine micelles. The equilibrium constants for the interaction of curcumin with egg and soy phosphatidylcholine were (3.26 +/- 0.2) x 10(5) and (2.64 +/- 0.2) x 10(5) M(-1), respectively. From the Scatchard plot of the fluorometric data, it was inferred that one molecule of curcumin could bind six molecules of phosphatidylcholine. The equilibrium constant for the phosphatidylcholine-curcumin interaction decreased with temperature, indicating the amphiphilic nature of curcumin. The DeltaG, DeltaH, and DeltaS values obtained for the interaction of egg phosphatidylcholine-curcumin were -7.8 +/- 0.3 kcal/mol, -9.6 +/- 0.4 kcal/mol, and -6.8 +/- 0.2 cal/mol/K, respectively. The fluorescence anisotropy measurements of curcumin with phosphatidylcholine suggested that the anisotropy of the curcumin molecule did not change in phosphatidylcholine. The interaction of divalent metal ions with phosphatidylcholine-curcumin in comparison with phosphatidylcholine-1-anilino-8-naphathalenesulfonic acid complex suggested the strong binding of curcumin to metal ions.

Change in the positional specificity of lipoxygenase 1 due to insertion of fatty acids into phosphatidylcholine deoxycholate mixed micelles.

Linoleic and arachidonic acids were inserted into phosphatidylcholine deoxycholate mixed micelles (PDM-micelles) with their tail groups buried inside and carboxylic groups exposed outside. The fatty acid hydrophobic tail had a high affinity for the hydrophobic region of phosphatidylcholine micelles. The fatty acids inserted into phosphatidylcholine micelles were better substrates for soybean lipoxygenase 1 (LOX1) with two distinct pH optima at 7.0 and 10.0. With Tween 20-solubilized linoleic acid, the enzyme had a pH optimum at 9.0, exclusively forming 13-hydroperoxides. However, with linoleic and arachidonic acids inserted into PDM-micelles, LOX1 synthesized exclusively 9- and 5-hydroperoxides, respectively. The enzyme brought about the transformation of the substrate either at pH 7.4 or at 10.0, less efficiently at pH 10.0. However, the regioselectivity of the enzyme was not altered by increasing the pH from 7.4 to 10.0. Thus, LOX1 could utilize fatty acids bound to membranes as physiological substrates. The enzyme utilized the carboxylic group of linoleic and arachidonic acids inserted into the PDM-micelles as a recognition site to convert the compounds into 9- and 5-hydroperoxides, respectively. This was confirmed by activity measurements using methyl linoleate as the substrate. Circular dichroism measurement of LOX1 with PDM-micelles suggested that while there was a small change in the tertiary structure of LOX1, the secondary structure was unaffected. Soybean LOX1, which is arachidonate 15-LOX, acted as "5-LOX", thus making it possible to change the regiospecificity of the LOX1-catalyzed reaction by altering the physical state of the substrate.

Inhibition of lipoxygenase 1 by phosphatidylcholine micelles-bound curcumin.

Curcumin (diferuloyl methane) from rhizomes of Curcuma longa L. binds to phosphatidylcholine (PC) micelles. The binding of curcumin with PC micelles was followed by fluorescence measurements. Curcumin emits at 490 nm with an excitation wavelength of 451 nm after binding to PC-mixed micelles stabilized with deoxycholate. Curcumin in aqueous solution does not inhibit dioxygenation of fatty acids by Lipoxygenase 1 (LOX1). But, when bound to PC micelles, it inhibits the oxidation of fatty acids. The present study has shown that 8.6 microM of curcumin bound to the PC micelles is required for 50% inhibition of linoleic acid peroxidation. Lineweaver-Burk plot analysis has indicated that curcumin is a competitive inhibitor of LOX1 with Ki of 1.7 microM for linoleic and 4.3 microM for arachidonic acids, respectively. Based on spectroscopic measurements, we conclude that the inhibition of LOX1 activity by curcumin can be due to binding to active center iron and curcumin after binding to the PC micelles acts as an inhibitor of LOX1.