CHARACTERIZATION OF DISULFIDE LINKAGES AND DISULFIDE BOND SCRAMBLING IN RECOMBINANT HUMAN MACROPHAGE-COLONY-STIMULATING FACTOR BY FAST-ATOM-BOMBARDMENT MASS-SPECTROMETRY OF ENZYMATIC DIGESTS

Fast-atom bombardment mass spectrometry was used to study disulfide bo nding patterns in heat-denatured human recombinant macrophage colony s timulating factor (rhM-CSF). The heat-denaturated protein was studied by analysis of the pattern of peptides in the proteolytic digests. Nat ive rhM-CSF is a homodimer with intramolecular disulfide linkages betw een Cys7-Cys90, Cys48-Cys139, and Cys102-Cys146 and intermolecular lin kages between Cys31-Cys31, and the pairs Cys157 and Cys159. Brief heat ing for 1 min leads to partial disulfide bond scrambling. In addition to the native disulfide bonds between Cys7-Cys90, Cys48-Cys139, and Cy s31-Cys31, nonnative disulfide bonds were detected between Cys48-Cys90 and Cys48-Cys102. When heated for 5 min the disulfide bonds of rhM-CS F are completely scrambled and lead to nonnative intramolecular disulf ide bonds between Cys48-Cys102 and Cys90-Cys102 and one intermolecular disulfide bond between Cys102-Cys102.