Journal article


Characterization of ascorbylated proteins by immunochemical methods


Publication Details
Author(s): Huber B, Pischetsrieder M
Publisher: American Chemical Society
Publication year: 1998
Volume: 46
Pages range: 3985-3990
ISSN: 0021-8561
Language: English

Abstract

L-Ascorbic acid (AA) binds covalently to proteins in a Maillard-type reaction (protein ascorbylation). An immunochemical screening method was developed to determine the products formed during protein ascorbylation. Thus, sheep serum albumin (SSA) was incubated with L-dehydroascorbic acid (DHA) to get highly modified ascorbylated SSA (DHA-SSA), and a polyclonal anti-DHA -protein antiserum was prepared using DHA-SSA as antigen. Noncompetitive and competitive ELISAs were performed to characterize the reactive epitopes. It was found that (1) the antibody binds to ascorbylated lysine but not to arginine or histidine residues; (2) the formation of reactive products is highly dependent on the presence of oxygen; (3) proteins that were glycated by sugars, such as glucose or ribose, show significant binding to the anti-DHA-protein antibody, indicating that the reactive epitope can also be generated by carbohydrates other than AA; (4) CML-protein [CML = Aε-(carboxymethyl)lysine] and CML can inhibit the antibody binding completely. These results show that the anti-DHA-protein antiserum consists mainly of anti-CML antibodies. Using specific anti-CML and anti-oxalic acid monoamide (OMA) antibodies (Pischetsrieder, M.; Larisch, B.; Seidel, W. J. Agric. Food Chem. 1997, 45, 2070-2075), it was found that both antisera bind significantly to DHA-proteins. Therefore, it can be concluded that CML is an important product formed during the reation of AA and proteins and that it is the main immunological epitope of ascorbylated protein. However, other predominant modifications on ascorbylated proteins besides CML and OMA cannot be excluded.



How to cite
APA: Huber, B., & Pischetsrieder, M. (1998). Characterization of ascorbylated proteins by immunochemical methods. Journal of Agricultural and Food Chemistry, 46, 3985-3990. https://dx.doi.org/10.1021/jf9803132

MLA: Huber, Birgit, and Monika Pischetsrieder. "Characterization of ascorbylated proteins by immunochemical methods." Journal of Agricultural and Food Chemistry 46 (1998): 3985-3990.

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