Analysis of disulphide linkages in bovine kappa-casein oligomers using two-dimensional electrophoresis.
Disulphide bonds play an important role in protein structure and function. Bovine kappa-casein (kappa-csn), an important glycoprotein in milk, contains two cysteines that can form disulphide bonds. On 2-D gels run under nonreducing conditions the kappa-csn in milk presented a complex pattern of monomers and disulphide-linked oligomers. Trains of spots corresponding to monomers to hexamers were observed as a result of the participation of different glycoforms and phosphoforms in oligomer formation. The dimers and trimers ran as doublets on the gel and analysis of the disulphide-linked peptides released from them after in-gel tryptic digestion showed they were the result of different disulphide linkages. The linkages were confirmed by MSMS. When milks with electrophoretically distinct genetic variants of kappa-csn were mixed and run on 2-D gels, they retained their distinct patterns indicating that disulphide exchange reactions or disulphide 'scrambling' was not occurring during 2-D analysis. The patterns observed represent the native distribution of kappa-csn in milk at harvest. The role and significance of the disulphide bonding of kappa-csn are discussed.
Holland
John W
JW
Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland, Australia. j.holland@imb.uq.edu.au
Deeth
Hilton C
HC
Alewood
Paul F
PF
D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000737:chemistry*; D002417:Cattle; D004220:Disulfides / Q000032:analysis*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D008892:Milk / Q000737:chemistry; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
D002364:Caseins; D004220:Disulfides; D020033:Protein Isoforms