Determination of the phosphorylation level and deamidation susceptibility of equine beta-casein |
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Authors: | Girardet Jean-Michel Miclo Laurent Florent Sabrina Mollé Daniel Gaillard Jean-Luc |
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Institution: | Laboratoire des BioSciences de l'Aliment, UC INRA 885, Faculté des Sciences et Techniques, Université Henri Poincaré-Nancy 1, Vandoeuvre-lès-Nancy, France. Jean-Michel.Girardet@scbiol.uhp-nancy.fr |
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Abstract: | beta-Casein was isolated from Haflinger mare's milk by RP-HPLC, and displayed microheterogeneity by urea-electrophoresis and 2-DE probably due to a variable degree of phosphorylation. To investigate the degree of phosphorylation, the primary structure of equine beta-casein was determined by tryptic hydrolysis and MS of peptides released and by MS of the protein treated by alkaline phosphatase. The molecular mass found for the apo-form of Haflinger mare's beta-casein (25 514 +/- 3 Da) was close to the theoretical mass of the reported sequence (GenBank AAG43954) modified by insertion of a region (residues 27-34) encoded by an exon sometimes out-spliced (25 511.40 Da). Hence, the beta-casein isolated from Haflinger mare's milk corresponded to a variant of 226 amino acid residues. The latter was composed by highly multi-phosphorylated isoforms with three to seven phosphate groups, and pIs, determined by 2-DE, ranging from 4.74 to 5.30. Moreover, the equine beta-casein was able to deamidate spontaneously, at the level of Asn in the potential deamidation motif (135)Asn-Gly(136). Approximately 80% of the protein was deamidated after 96 h of incubation under physiological conditions. |
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Keywords: | β‐Casein 2‐DE Equine milk Phosphorylation Spontaneous deamidation |
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