Multiple forms of equine α-lactalbumin:: evidence for N-glycosylated and deamidated forms

Equine raw milk whey proteins were separated by anion-exchange fast protein liquid chromatography and characterised by alkaline polyacrylamide get electrophoresis (PAGE), sodium dodecyl sulphate PAGE, and bi-dimensional PAGE. Approximately, 1% of alpha-lactalbumin (alpha-LA) was N-glycosylated. A minor N-glycosylated form of lysozyme was also found in equine milk. On the other hand, two non-glycosylated a-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 a-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 alpha-LA at 37degreesC. The initial rate of this chemical degradation was 4.5 mum ammonia liberated per hour, in 150 mm sodium phosphate buffer pH 7.4 at 37degreesC. Deamidation induced a slight variation in secondary structure content, but no significant change in the tertiary structure of the equine alpha-LA was studied by circular dichroism in the near- and far-UV regions. (C) 2003 Elsevier Ltd. Ail rights reserved.