Alkaline cleavage of covalent bonds in chicken insulin and bovine alpha-lactalbumin analyzed by matrix-assisted laser desorption/ionization-mass spectrometry.
In the course of searching methods to extract proteins from Coomassie blue-stained polyacrylamide gels, we found proteins are extracted in relatively high recovery when the gel pieces are soaked in alkaline solutions. However, alkaline conditions are known to cause decomposition of proteins, especially peptide bond cleavage and disulfide degradation. We studied the effects of alkaline on two purified proteins, chicken insulin and bovine alpha-lactalbumin, both containing four disulfide bonds in their structure. The process of covalent bond cleavage was traced by analyzing the mass spectra of the proteins using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). When the proteins are kept at pH 13 in the presence of 0.1% dithithreitol (DTT), peptide bonds at the C-terminal side of asparaginyl residues are preferably cleaved producing succinimides, whereas cysteinyl residues are not decomposed. In the absence of DTT, the disulfide bonds of the proteins are decomposed by alkaline and the cleavage of the peptide bonds are less obvious, possibly because the conformation of the proteins are partially retained until the full decomposition of disulfide bonds. These results identified for the first time the cleavage sites of proteins under alkaline treatment and further suggested the general tendency of the reactions, both in the presence and absence of DTT.
Manabe
Takashi
T
Department of Chemistry, Faculty of Science, Ehime University, Matsuyama, Japan. manabet@dpc.ehime-u.ac.jp
Jin
Ya
Y
D000468:Alkalies / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002645:Chickens; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000737:chemistry*; D007768:Lactalbumin / Q000737:chemistry*; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000468:Alkalies; D004220:Disulfides; D007328:Insulin; D007768:Lactalbumin; D009113:Muramidase; D004229:Dithiothreitol