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.
Melarsen oxide [p-(4,6-diamino-1,3,5-triazin-2-yl)aminophenylarsonous acid (MEL)], which selectively bridges spatially neighboring bis-cysteinyl residues in (reduced) proteins, was used to trap folding intermediates chemically during 1) time-dependent renaturation of recombinant human macrophage colony-stimulating factor (rhM-CSF); by redox refolding in vitro; 2) reductive unfolding in the presence of the trapping reagent; and 3) denaturing unfolding reactions in urea and guanidinium hydrochloride. Characterization of intermediates from folding and unfolding reactions was performed by electrospray ionization mass spectometry (ESI-MS). In all folding and unfolding reactions a characteristic dimeric intermediate with two attached melarsen oxide (MEL) groups was observed, suggesting that these rhM-CSF beta species were important refolding intermediates. These intermediates presented a characteristic "charge structure" in ESI spectra with a most abundant 26+ charged molecular ion whereas the mature homodimeric rhM-CSF beta showed a most abundant 23+ molecular ion, indicating that the final product was more compact. The major locations of the two MEL groups were identified by mass spectrometric peptide mapping at cysteine residues C157 and C159 from each monomer. Cysteine residues C7 and C90 were minor modification sites. The mass spectrometric results from the in vitro folding reactions of rhM-CSF beta are in agreement with intrinsic tryptophan fluorescence measurements and are consistent with the folding pathway that starts with a fully reduced monomer (R), includes partially folded monomeric intermediates (M) and dimeric intermediates (D), and yields a final product with the native tertiary structure (N): 2R ==> 2M ==> D ==> N. Our results show that selective chemical trapping of bis-thiol groups of proteins with MEL permits study of folding pathways by mass spectrometric structure characterization of intermediates with otherwise transient conformations.
Reverse-phase high-performance liquid chromatography has been used to fractionate ribosomal proteins from Escherichia coli and rabbit reticulocytes. Different column packing materials and solvent systems were compared for their effectiveness with bacterial proteins. A large-pore (300 A) short alkyl chain support (Altex RPSC) in conjunction with a triethylamine phosphate (pH 2.2)/acetonitrile solvent system was particularly effective and separated mixtures of total protein from each ribosomal subunit into a number of peaks approaching the actual number of proteins present. For example, with the use of the Altex RPSC column, the 21 proteins of 30S subunits were resolved into 18 distinct peaks, and the 33 proteins of the 50S subunits were resolved into 28 peaks. Overall recovery varied from 75% to 90% in different experiments. The composition of each peak was established by two-dimensional gel electrophoresis. Relatively acidic proteins, for example, S1 and L7/L12 of Escherichia coli, were bound more tightly to the column and recovered in lower yields than the other more basic proteins. Proteins that were incompletely resolved in a single step could be obtained in pure form by rechromatography on the same column with an altered gradient or with a different type of reverse-phase packing material. Ribosomal proteins from rabbit reticulocytes were also separated with good resolution and yield by using the RPSC column.
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