Purification and Partial Characterisation of Glycosylated Bovine Alpha Lactalbumin

Abstract

Bovine alpha lactalbumin exists in four different forms. These are F, M, S, and S,' so named because of relative position on non-denaturing polysaccharide gels. F, S, and S, are minor components that make up 15% ofthe total alpha lactalbumin and S, and S' have shown to be glycoforms of protein. The lectin affinity column chromatography (Concanvalin A ) was used to seprate glycosylated protein from non glycosyalated contaminants from acid whey. Size exclusion chromatography was then used to separate the glycosylated alpha lactalbumin from the immunoglobulins and other contamnants. In order to analyse the number of oligosaccharide chains bound to protein, they were enzymatically cleaved from the protein using peptide-N4-(N-acetyl-(3-glucosamnyl) asparagines aminase (PNGase F), then separated using high pH anion exchange chromatography with pulsed amperometic detection (HPEA/PAD) and tluorophore assisted carbohydrate electrophoresis (FACE). Electrospray mass spectroscopy was used to confirm the results of these techniques. The structure of the main sialic acid containing glycan and neural glycan wete postulated, which represent S, and S, respectively. It is interesting to note that these molecules differs only one sialic acid residue.