Abstract
Purpose. We examined the metal-catalyzed oxidation of brain-derivedneurotrophic factor (BDNF) using the Cu(II)/ascorbate/O2 modeloxidative system.
Methods. Electrospray ionization mass spectrometry, peptide mappingand amino acid analysis were utilized to determine the nature of thecovalent modification induced by the metal-catalyzed oxidative system.Additionally, analytical ultracentrifugation, the Bradford assay, circulardichroism and ANSA dye-binding were used to determine the natureof any conformational changes induced by the oxidation.
Results. Exposure of BDNF to the Cu(II)/ascorbate/O2 system led tothe modification of ca. 35% of Met92 to its sulfoxide, and to subsequentconformational changes. The proteolytic digestion procedure wassensitive to this conformational change, and was unable to detect themodification. Chemical digestion with CNBr, however, was not sensitive tothis change, and allowed for the identification of the site ofmodification.
Conclusions. The modification of Met92 to its sulfoxide rendered theoxidized BDNF inaccessible to proteolytic digestion, due toconformational changes associated with the oxidation.
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Jensen, J.L., Kolvenbach, C., Roy, S. et al. Metal-Catalyzed Oxidation of Brain-Derived Neurotrophic Factor (BDNF): Analytical Challenges for the Identification of Modified Sites. Pharm Res 17, 190–196 (2000). https://doi.org/10.1023/A:1007569431038
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DOI: https://doi.org/10.1023/A:1007569431038