Effects of reaction mixture and other components on the determination of the equilibrium and rate constants of the hydration reactions of anthocyanins

The equilibrium and rate constants of the hydration and deprotonation reactions of anthocyanins show how their color intensity changes with pH. In the cases of several anthocyanins, the constants for each obtained by several methods are different. In an effort to resolve these discrepancies, we have examined the effects of several components of the pH-jump experiments on the values of the constants. Storage of the buffers to be used in pH-jump experiments in Pyrex or borosilicate glass bottles results in increasing Al3+ concentration in the buffers over several weeks. When these buffers are used, the anthocyanins with two OH groups on the B ring complex with the Al3+ which leads to major changes in their spectra, in the equilibrium position, and in the apparent first-order rate constant. Thus, constants determined on the same anthocyanin using the same buffers stored in glass bottles may be different at different times. During the reduction of the experimental data to the rate and equilibrium constants, two divergences from the expected behavior were found. In the calculation Ka + Kh for the anthocyanin acylated with 4-hydroxy-3,5-dimethoxycinnamic acid (6-O-(4-hydroxy-3,5-dimethoxycinnamoyl)-β-d-glucopyranosyl-(1 → 6)-[β-d-xylopyranosyl-(1 → 2)]-β-d-galactopyranosyl-(1 → O3)-cyanidin), the plotted points appear to fit two straight lines, intersecting at an equilibrium pH near pH 4. In the calculation which leads to the individual constants of both anthocyanins examined here, the points below an equilibrium pH of pH 4 curve upward from the line that describes the points from an equilibrium pH above 4. Differences in the composition of solutions used in pH-jump experiments examined here, include (1) the addition of phosphate to the acetate buffer, (2) the presence of 0.5 M NaCl, and (3) the solution of the anthocyanin in either 0.1 N HCl or 0.1 N HOAc. These changes gave differences that were statistically significant in some of the constants for each of the two anthocyanins examined. The constants were both qualitatively and quantitatively different.