The pH equilibria and the zinc ion and bovine serum albumin (BSA) binding behavior of curcumin and two chemically modified curcumins (CMCs), namely 4-methoxycarbonylcurcumin (CMC 2.5) and 4-phenylaminocarbonyl bis-demethoxy curcumin (CMC 2.24), were studied, in order to understand the basis of their differential effects on the zinc-enzyme matrix metalloproteinases (MMPs) as well as the effect of charge state on their behavior in vivo. Moreover, all three compounds transform rapidly in the pH range 5-10, CMC 2.5 largely in one step, and CMC 2.24 and curcumin first in a rapid process to an intermediate form that still displays an enolic and two phenolic hydrogen-ion equilibria, and then more slowly to forms absorbing primarily in the lower UV and lacking the strong absorbance in the visible characteristic of the enol-centered chromophore. The binding of these compounds in one of the hydrophobic pockets of the major transport protein, serum albumin, was therefore studied. CMC 2.24 binds more strongly to BSA than curcumin, with a dissociation constant of 0.56±0.08 μM compared to 1.32±0.17 μM. Binding to BSA shifts the decomposition half-lives from tens of seconds to tens of hours. The zero-time acid dissociation constants (pKa) for species H3D, H2D-, and HD2- are 8.41, 9.94 and 11.2; 6.98, 8.40 and 9.8; and 6.50 and 8.82 ; for curcumin, CMC 2.24, and CMC 2.5 respectively (there is no distinguishable pKa3 for CMC 2.5). Zn2+ binds most strongly to CMC 2.24 compared to CMC 2.5 and curcumin, with dissociation constants of 0.77±0.02, 1.88±0.07, and 1.39±0.09 mM. The increased acidity and Zn2+ and BSA affinities of CMC 2.24 correlate with its greater biological activity.
Keywords: Matrix metalloproteinase, curcumin, pKa, zinc-binding, bovine serum albumin binding, time-dependence, UV-visible absorbance and equilibria, fluorescence quenching, Förster energy transfer