Selective bridging of bis-cysteinyl residues by arsonous acid derivatives as an approach to the characterization of protein tertiary structures and folding pathways by mass spectrometry.

Bis-cysteine selective modifications were successfully applied with melarsen oxide (MEL), an arsonous acid derivative, for tertiary structural studies of peptides and a model protein. The arsonous acid modified peptides and proteins were amenable to direct characterizations by mass spectrometry, e.g.

Characterization of the folding pathway of recombinant human macrophage-colony stimulating-factor beta (rhM-CSF beta) by bis-cysteinyl modification and mass spectrometry.

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.

Biochemical characterization and mass spectrometric disulfide bond mapping of periplasmic alpha-amylase MalS of Escherichia coli.

Periplasmic alpha-amylase of Escherichia coli, the malS gene product, hydrolyzes linear maltodextrins. The purified enzyme exhibited a Km of 49 microM and a Vmax of 0.36 micromol of p-nitrophenylhexaoside hydrolyzed per min per mg of protein.