Preparative separation of small molecular weight peptides from casein hydrolysate using gel filtration and immobilized metal ion affinity chromatography.
A two step method consisting of a gel filtration step, followed by a Immobilized Metal Affinity Chromatography (IMAC) step using a IDA-Cu coupled Sephadex G-25 column, on a preparative scale is described for the group separation of peptides from a casein hydrolysate. The 48 groups of peptides thus separated are further characterised by RP-HPLC and amino acid analysis. Some peptides after the analytical RP-HPLC step are further characterised by sequencing. An insight into the mechanism of retention on IMAC of the peptides is attempted. In such complex mixtures as casein hydrolysate, the peptide-peptide interaction can mask the potential sites of interactions in a single peptide. The results obtained using volatile buffers as eluents show the possibility of using IMAC step as an alternative to obtain gram quantities of group of peptides free of salts from complex protein hydrolysates.
Gauthier
J
J
Département de Sciences et Technologie des Aliments, Université Laval, Sté-Foy, Québec.
Amiot
J
J
Vijayalakshmi
M A
MA
D000595:Amino Acid Sequence; D002364:Caseins / Q000032:analysis*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000302:isolation & purification*; D011492:Protein Hydrolysates / Q000032:analysis*
D002364:Caseins; D010455:Peptides; D011492:Protein Hydrolysates; C026375:trypticase-soy broth