Enrichment and functional characterization of copper-binding peptides from food hydrolysates.

Imbalances in cellular copper are increasingly implicated in metabolic disorders. Food-derived peptides are gaining attention for their ability to alleviate metabolic disease symptoms with little to no toxicity. In this work, we enriched copper-binding peptides from enzymatic digestions of rice bran protein hydrolysates via Cu(ii)-based immobilized-metal affinity-based separations, identified the sequences by mass spectrometry, and performed physicochemical and sequence analysis of the enriched peptides. A subset of the enriched peptides representing a range of sequences and hydrophobicities were synthesized and individually assessed further for their solution-based Cu(ii) reactivity and cellular activity. Among the peptides tested, Cu(ii)-binding was most potent with those containing a histidine as the second residue, reminiscent of pseudo-ATCUN motifs. Relative Cu(ii)-binding affinity showed good correlation with the ability of the peptides to reduce Cu(ii) reactivity, as measured with a reaction-based probe, while discrepancies in trends were observed in the scavenging activity of Cu(ii)-generated ROS. To test the hypothesis that solution-based Cu(ii)-binding properties predict cellular bioactivity, we assessed whether the individual Cu(ii)-binding peptides could recapitulate the previously reported AMPK activation in hepatocytes by whole rice bran hydrolysates. This represents a critical validation step for using in vitro metal-binding assays to predict biological function. While the majority of the peptides affected AMPK activation, the degree and direction varied in ways that Cu(ii) chelation could not explain, suggesting that the beneficial behavior of the hydrolysates cannot be simplified to its in vitro Cu(ii) reactivity. Nonetheless, we demonstrate a strategy for identifying putative metal-binding peptides from complex food-derived mixtures and characterizing their chemical and biological activity.