| Protein-bound forms of tannin have not been tested as radical scavengers although this is their likely form in vivo. The 2,2 ′ azinobis(3-ethylbenzothiazoline 6-sulfonic acid) radical cation (ABTS.+) decolorization assay was used to determine the antioxidant activity of the polyphenol epicatechin16 (4 → 8) catechin (procyanidin, PC) alone or in complex with the model proteins bovine serum albumin (BSA) or gelatin. PC had a molar antioxidant capacity of approximately 54, 92 or 108 radicals at pH values of 3.0, 4.9 or 7.4 respectively. Radical scavenging occurred via a rapid step followed by a slow step. Interaction with gelatin reduced the rapid scavenging step by 50% (PC-BSA mixtures reduced by 15%). Inhibition corresponded to the prevalence of precipitable PC-protein complexes. Surprisingly, inhibition was not detected after 10 minutes. Reaction with ABTS.+ converted the PC-protein precipitates from a dissociable to a refractory form, a process I call a radical sink. Since tannins bind selectively to proline-rich proteins (PRPs) under non-oxidizing conditions, I investigated whether the radical sink displayed similar selectivity. Bovine serum albumin (BSA) was used as a target at pH 4.9 and chymotrypsinogen (CT) at pH 7.4. Sodium dodecyl-sulfate polyacrylamide gel-electrophoresis was used to quantify how much target remained soluble after target was mixed with PC in the presence or absence of oxidizing stress ABTS.+ and with or without competing protein. In the absence of competitor, oxidation caused a straightforward conversion of dissociable BSA-PC precipitate to a refractory form. For CT-PC approximately twice as much CT-PC was refractory as was precipitated as a dissociable complex in the non-oxidized case. Gelatin and human salivary PRP prevented BSA precipitation and protected BSA from refractory conversion. Salivary PRP prevented CT precipitation and fully protected CT against refractory conversion. Although BSA and gelatin easily prevented CT from precipitating they only provided minimal protection for CT under oxidizing conditions. Thus, a competitor's ability to prevent target precipitation does not always correspond to the selectivity of the radical sink for that competitor. In summary, PC is a high capacity, protein-selective radical scavenger and PC-protein complexes might serve as effective radical sinks within the digestive tract. |
