Multiple forms of equine α-lactalbumin: evidence for N-glycosylated and deamidated forms
2004
Girardet, J.M. | N'negue, M.A. | Egito, A.S. | Campagna, S. | Lagrange, A. | Gaillard, J.L.
Equine raw milk whey proteins were separated by anion-exchange fast protein liquid chromatography and characterised by alkaline polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulphate PAGE, and bi-dimensional PAGE. Approximately, 1% of α-lactalbumin (α-LA) was N-glycosylated. A minor N-glycosylated form of lysozyme was also found in equine milk. On the other hand, two non-glycosylated α-LA isoforms with similar molecular masses (14,215±4 Da) were shown to be present. Their respective apparent isoelectric points were 5.25 and 4.94. These isoforms did not correspond to different genetic variants and were not the result of α-LA modulation by calcium ions. They corresponded rather to a non-enzymatic deamidation process of a single asparagine side-chain, the most acidic isoform being spontaneously generated from the less acidic isoform by simple incubation of α-LA at 37°C. The initial rate of this chemical degradation was 4.5 μm ammonia liberated per hour, in 150 mm sodium phosphate buffer pH 7.4 at 37°C. Deamidation induced a slight variation in secondary structure content, but no significant change in the tertiary structure of the equine α-LA was studied by circular dichroism in the near- and far-UV regions.
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