Disulfide linkages in the in vitro refolded intermediates of recombinant human macrophage-colony-stimulating factor: analysis of the sulfhydryl alkylation of free cysteine residues by fast-atom bombardment mass spectrometry.
Fast-atom bombardment mass spectrometry was used to follow the time course of disulfide bond formation during in vitro refolding of recombinant human macrophage-colony-stimulating factor. The content of iodoacetamide-alkylated half-cystines in proteolytic peptides of trapped refolding intermediates collected at 0, 6, 17, 24, and 72 hr was determined under reducing conditions. Size-exclusion high-performance liquid chromatography analyses of the collected alkylated samples indicate that aggregated monomer proceeded through a nonaggregated monomer to an intermediate dimer and finally to the fully folded and active dimer. Underalkylation was first detected by fast-atom bombardment mass spectrometry in 17-hr samples at Cys157 and Cys159 and this corresponded to the first sample containing dimer. Analyses of intermediates from subsequent time points indicated a decrease in alkylated sulfhydryls, and at 72 hr no alkylated peptide was detected. Early samples containing only monomer showed no evidence of disulfide bonds, and the occurrence of disulfide shuffling at the monomer stage could be ruled out under the highly reducing conditions used for refolding. Biological activity was not detectable in early samples but increased to 3.6% after 24 hr of refolding and to 86% of maximum at the 72-hr time point.
Department of Agricultural Chemistry, Oregon State University, Corvallis 97331.
M O
MO
Glocker
B
B
Arbogast
R
R
Milley
C
C
Cowgill
M L
ML
Deinzer
D000478:Alkylation; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D004220:Disulfides / Q000032:analysis*; D006801:Humans; D016173:Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016339:Spectrometry, Mass, Fast Atom Bombardment / Q000379:methods
D004220:Disulfides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D016173:Macrophage Colony-Stimulating Factor; D003488:Cyanogen Bromide