title: A new look on protein-polyphenol complexation during honey storage: is this a random or organized event with the help of dirigent-like proteins?
abstract: Honey storage initiates melanoidin formation that involves a cascade of seemingly unguided redox reactions between amino acids/proteins, reducing sugars and polyphenols. In the process, high molecular weight protein-polyphenol complexes are formed, but the mechanism involved remains unknown. The objective of this study was twofold: to determine quantitative and qualitative changes in proteins in honeys stored for prolonged times and in different temperatures and to relate these changes to the formation of protein-polyphenol complexes. Six -month storage decreased the protein content by 46.7% in all tested honeys (t-test, p<0.002) with the rapid reduction occurring during the first three month. The changes in protein levels coincided with alterations in molecular size and net charge of proteins on SDS -PAGE. Electro-blotted proteins reacted with a quinone-specific nitro blue tetrazolium (NBT) on nitrocellulose membranes indicating that quinones derived from oxidized polyphenols formed covalent bonds with proteins. Protein-polyphenol complexes isolated by size-exclusion chromatography differed in size and stoichiometry and fall into two categories: (a) high molecular weight complexes (230-180 kDa) enriched in proteins but possessing a limited reducing activity toward the NBT and (b) lower molecular size complexes (110-85 kDa) enriched in polyphenols but strongly reducing the dye. The variable stoichiometry suggest that the large, "protein-type" complexes were formed by protein cross-linking, while in the smaller, "polyphenol-type" complexes polyphenols were first polymerized prior to protein binding. Quinones preferentially bound a 31 kDa protein which, by the electrospray quadrupole time of flight mass spectrometry (ESI-Qtof-MS) analysis, showed homology to dirigent-like proteins known for assisting in radical coupling and polymerization of phenolic compounds. These findings provide a new look on protein-polyphenol interaction in honey where the reaction of quinones with proteins and polyphenols could possibly be under assumed guidance of dirigent proteins.
authors.affiliation: Department of Drug Discovery and Development, Bee-Biomedicals Inc., St. Catharines, Ontario, Canada ; Department of Biological Sciences, Brock University, St. Catharines, Ontario, Canada.
authors.forename: Katrina
authors.initials: K
authors.lastname: Brudzynski
authors.forename: Calvin
authors.initials: C
authors.lastname: Sjaarda
authors.forename: Liset
authors.initials: L
authors.lastname: Maldonado-Alvarez
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D006722:Honey / Q000032:analysis*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D059808:Polyphenols / Q000378:metabolism*; D011309:Preservation, Biological; D011809:Quinones / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013194:Staining and Labeling; D013696:Temperature; D013778:Tetrazolium Salts / Q000378:metabolism
chemical_list: D010455:Peptides; D010940:Plant Proteins; D059808:Polyphenols; D011809:Quinones; D013778:Tetrazolium Salts
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