Structural and Molecular Basis of Interaction of Curcumin with Proteins

Curcumin (diferuloylmethane), a natural lipid-soluble yellow compound from
the plant Curcuma longa L., is a potent antioxidant, antitumorigenic and antiinflammatory
molecule. Very limited solubility and stability in aqueous medium
and poor bioavailability limits the use of curcumin as an efficient therapeutic
agent. The present study was focused, towards exploring possible
medium/carrier to increase the solubility and stability of curcumin. Further,
studies were carried out to understand the basis of interaction.
b-Lactoglobulin (bLG), the major whey protein, can solubilize and bind many
small hydrophobic molecules. The stability of curcumin bound to bLG in
solution was enhanced 6.7 times, in comparison to curcumin alone, in aqueous
solution. The complex formation of curcumin with bLG has been investigated
employing spectroscopic techniques. bLG interacts with curcumin at pH 7.0,
with an association constant of 1.04 ± 0.1 ´ 105 M-1, to form a 1:1 complex, at 25
°C. Entropy and free energy changes for the interaction, derived from the van’t
Hoff plot, were 18.7 cal mol-1 and -6.8 kcal mol-1, respectively; the interaction is
hydrophobic in nature. Interaction of bLG with curcumin does not affect either
the conformation or the state of association of βLG. Binding studies with
denatured βLG, effect of pH on curcumin-bLG interaction, Förster energy
transfer measurements and molecular docking studies suggested that curcumin
binds to the central calyx of bLG. Nanoparticles of bLG, prepared by desolvation,
are found to encapsulate curcumin with > 96% efficiency. The solubility of
3
curcumin in βLG nanoparticle, significantly enhanced to ~ 625 ?M in comparison
with its aqueous solubility (30 nM).
The stability of curcumin in the presence of aS1-casein was enhanced by ~ 39
folds at pH 7.2, in comparison of the stability of curcumin in aqueous medium.
Curcumin binds to aS1-casein at two different binding sites, one with high
affinity and another with low affinity characterized by association constants of
2.01 ± 0.6 ´ 106 and 6.3 ± 0.4 ´ 104 M-1, respectively. The carboxyl-terminal of aS1-
casein (100-199 residues) and the residues 14-24 in aS1-casein are hydrophobic in
nature. The free energy change for the binding of curcumin to aS1-casein, BG° at
27 °C, was -8.65 kcal mol-1. The BH° and BS° for the binding reaction were
estimated to be -1.28 kcal mol-1 and 24.7 cal mol-1, respectively. Hydrophobic
force was the main contributing factor for the interaction of curcumin to aS1-
casein. Chaperone activity of aS1-casein was enhanced when bound to curcumin.
The biological activity of curcumin, like its protection against hemolysis was
unchanged on interaction with aS1-casein. Average size of curcumin
encapsulated aS1-casein nanoparticles was ~ 166 ± 5 nm. Encapsulation efficiency
of curcumin was > 94% and 38% curcumin release was observed from
nanoparticles in 24 h. The solubility of curcumin in aS1-casein nanoparticle was
enhanced to ~ 620 ?M in comparison with its aqueous solubility (30 nM).
The ability of curcumin to measure the surface hydrophobicity of proteins was
analyzed and compared with a standard fluorescent probe - cis-parinaric acid.
4
Surface hydrophobicity of - BSA, βLG, soy LOX-1, ovalbumin and lysozyme are
in the order BSA > βLG > soy LOX-1 > ovalbumin > lysozyme. The binding
affinities of curcumin decreased with the decrease in surface hydrophobicity of
proteins. Surface hydrophobicity index value (S0), determined using curcumin
correlated with the S0 values of protein calculated using CPA. The S0 value of
proteins determined using curcumin decreased in the presence of urea,
suggesting the possible use of curcumin as a probe to determine the surface
hydrophobicity of proteins. Because of low quantum yield of curcumin
compared to ANS and CPA, it may not be an appropriate fluorescent probe for
measuring the S0 of proteins.
The structural similarities between CPA and linoleic acid, the substrate for soy
LOX-1, instigated to study the inhibition of soy LOX-1 with CPA. CPA, a C18
fatty acid inhibits soy LOX-1 activity with the IC50 value of 18.8 mM. The
mechanism of inhibition of soy LOX-1 by CPA was competitive with the Ki value
of 9.8 mM. CPA binds close to iron cofactor with the distance of carboxylate
group of CPA to the iron being 3.3 Å. The binding constant for the binding of
CPA to soy LOX-1 is 2.1 ± 0.5 ´ 104 M-1.
Tetrahydrocurcumin (THC; 1,7- bis(4-hydroxy-3-methoxyphenyl)heptane-3,5-
dione) – the reduced form of curcumin, is the major metabolite in vivo. Inhibition
of soy LOX-1 by THC was studied and the kinetics and mechanism of inhibition
was evaluated. THC inhibited soy LOX-1 activity with an IC50 value of 59.4 mM
for THC in aqueous solution and 44.6 mM for PC micelles encapsulated THC. The
5
lag phase for enzyme activation from its resting state increased with increasing
concentrations of THC. A mixed linear type of inhibition of LOX-1 was observed
with a Ki value of 39 mM. Molecular docking simulations suggested the binding
of THC near the iron cofactor. Spectroscopic and CD studies revealed that, THC
could prevent the conversion of the resting inactive ferrous form of the enzyme
to its active ferric form thus inhibiting the enzyme. From these studies, it can be
concluded that THC is less powerful in inhibiting the LOX-1 in comparison to
curcumin however, the higher solubility and stability of THC, compared to
curcumin, provides valuable leads for the use of this compound as an alternative
to curcumin in anti-inflammatory drugs. These observations indicate the
importance of methylene bridge at carbon seven in curcumin, in inhibiting the
soy LOX-1 enzyme.
Carbonic anhydrase is a family of metalloenzymes that catalyze the rapid
conversion of carbon dioxide to bicarbonate and protons, and is involved in
biomineralization process. It catalyzes the reversible hydration of CO2 to HCO3-
and H+. It is a zinc containing enzyme with the molecular weight of 29000 Da
and in red blood cells, it facilitates the transportation of CO2 out of the body. The
effect of curcumin on the activity of carbonic anhydrase was studied. No change
in the activity of carbonic anhydrase, in vitro in the presence of curcumin was
observed. Though, curcumin is a known chelator of metal ions, it had no effect
on redox inactive metal - zinc containing enzyme.