Eliminating disulfide exchange during glutamyl endopeptidase digestion of native protein.
Numerous advantages of using immobilized enzymes over free-solution protein digests have been cited in the literature. This investigation examines both the rate of hydrolysis and the extent of disulfide bond exchange in disulfide bridged dipeptide fragments formed during proteolysis of native protein. Glutamyl endopeptidase as both an immobilized enzyme and in free solution was used in these studies. It was found that extensive hydrolysis of insulin was achieved in 2 min with immobilized enzyme cartridges operated in the stopped-flow mode orders. This is orders of magnitude faster than was seen in free solution. Other advantages ranging from ease of use and reduction in sample size to the potential for automation were also noted with the immobilized enzyme cartridge. Normal free-solution proteolysis generally requires 12-24 h, based on the lower enzyme-to-substrate ratio in solution. A disturbing feature noted in these lengthy free-solution reactions was the tendency to form disulfide bridged peptide artifacts. This could lead to the erroneous conclusion that disulfide bonding in a sample was not that of the native protein. It is concluded that the advantage of immobilized enzymes over free-solution reactions will be most important in the pharmaceutical industry where proteolytic fragment "fingerprinting" of recombinant proteins is being used to confirm structure.
Dormady
S J
SJ
Department of Chemistry, Purdue University, Lafayette, IN 47907, USA.
Lei
J
J
Regnier
F E
FE
D000595:Amino Acid Sequence; D004220:Disulfides / Q000737:chemistry*; D019075:Electrophoresis, Capillary; D006801:Humans; D006868:Hydrolysis; D007328:Insulin / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004220:Disulfides; D007328:Insulin; D010446:Peptide Fragments; D011506:Proteins; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase