Covalent dimerization of recombinant human interferon-gamma.
An apparently nonreducible covalent dimer has been consistently observed as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These studies endeavor to understand the nature of this covalent dimerization. Amino acid sequence analysis of a recombinant human interferon-gamma stored for 2 years showed a major sequence starting with the intact N-terminal methionine and a minor sequence corresponding to the C-terminal seven amino acids of the intact protein. Moreover, when the same material was analyzed by gel filtration in the presence of 8 M urea, a minor peak corresponding to the dimer was observed prior to the monomer peak. Reducing and nonreducing SDS-PAGE also showed a minor band corresponding to a dimer. These results suggest that the reactions of C-terminal processing and dimerization have occurred during storage. Reverse-phase chromatography of stored, unfractionated material showed three peaks. Mass spectral analysis of the first, second, and third peaks gave molecular weights of 16,900, 16,100, and 33,000. Since no major cleavage was observed in the N-terminal region of the protein, the observed masses suggest that the first peak corresponds to residues 1 to 144 (a full-length molecule), the second peak to residues 1 to 137 (des 7 interferon-gamma), and the third peak to a dimer. Calculation of theoretical molecular weight from the amino acid sequence suggests that this dimer corresponds to some combination of the intact protein and des 7 protein. Tryptic peptide maps in conjunction with sequence and mass analyses identified a new tryptic peptide in the map of the dimer corresponding to residue 133 to 137 followed by residues 1 to 7. The conclusion is recombinant methionyl human interferon-gamma undergoes a specific cleavage at the C-terminal side of residue 137 phenylalanine, and a conventional peptide bond was formed between residues 137 and 1 methionine.
Lauren
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SL
Amgen Inc., Amgen Center, Thousand Oaks, California 91320-1789.
Arakawa
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T
Stoney
K
K
Rohde
M F
MF
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry* / Q000378:metabolism; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D011994:Recombinant Proteins; D014357:Trypsin
D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D007371:Interferon-gamma; D014357:Trypsin