Residual metals cause variability in methionine oxidation measurements in protein pharmaceuticals using LC-UV/MS peptide mapping.

Methionine oxidation has been demonstrated to play an important role in protein stability in vitro and in vivo. It may also cause changes in biological activity and immunogenicity profile of therapeutic proteins. Therefore, it is critical to monitor methionine oxidation in biopharmaceuticals during process and formulation development, as well as long-term stability studies.

Spectroscopic and computational studies of Ni superoxide dismutase: electronic structure contributions to enzymatic function.

Ni-containing superoxide dismutase (NiSOD) is the most recently discovered member of the class of metalloenzymes that detoxify the superoxide radical in aerobic organisms. In this study, we have employed a variety of spectroscopic and computational methods to probe the electronic structure of the NiSOD active site in both its oxidized (NiSOD(ox), possessing a low-spin (S = (1)/(2)) Ni(3+) center) and reduced (NiSOD(red), containing a diamagnetic Ni(2+) center) states. Our experimentally validated computed electronic-structure description for NiSOD(ox) reveals strong sigma-bonding interactions between Ni and the equatorial S/N ligands, which give rise to intense charge-transfer transitions in the near-UV region of the absorption spectrum.

S K-edge X-ray absorption spectroscopic investigation of the Ni-containing superoxide dismutase active site: new structural insight into the mechanism.

Superoxide dismutases protect cells from the toxic effects of reactive oxygen species derived from superoxide. Nickel-containing superoxide dismutases (NiSOD), found in Streptomyces species and in cyanobacteria, are distinct from Mn-, Fe-, or Cu/Zn-containing SODs in amino acid sequence and metal ligand environment. Sulfur K-edge X-ray absorption spectroscopic investigations were carried out for a series of mono- and binuclear Ni model compounds with varying sulfur ligation, and for oxidized and reduced NiSOD to elucidate the types of Ni-S interactions found in the two oxidation states.

Expression, reconstitution, and mutation of recombinant Streptomycescoelicolor NiSOD.

Nickel-dependent superoxide dismutases (NiSODs) represent a novel solution to controlling the deleterious effects of reactive oxygen species derived from superoxide in biology. The expression of recombinant Streptomyces coelicolor NiSOD and its in vitro processing and reconstitution to yield fully active enzyme is reported. The results of studies of NiSODs involving mutations in two putative nickel binding ligands are also reported.

Inner-sphere complexation of cobalt(II) 2,9-dimethyl-1,10-phenanthroline ([Co(neo)]2+) with commercial and sol-gel derived silica gel surfaces.

[Co(2,9-dimethyl-1,10-phenanthroline)(solvent)4]2+ ([Co(neo)]2+) undergoes a significant decrease in symmetry to form an inner-sphere surface complex when grafted directly on performed silica or introduced during the sol-gel process. The visible and X-ray absorption spectra of the surface adducts are interpreted in terms of a binding mode in which the Co(II) center has a highly distorted pseudo-C2v symmetry. The interaction of [Co(neo)]2+ with the silica surface was analyzed using an acid-base equilibrium relationship.

Pulsed ENDOR and ESEEM Study of [Bis(maleonitriledithiolato)nickel](-): An Investigation into the Ligand Electronic Structure.

By a combination of Q-band pulsed ENDOR (electron nuclear double resonance) and X-band ESEEM (electron stimulated echo envelope modulation) techniques, we have determined the hyperfine tensors for ethylene (C1) and cyano (C2) carbons and N, of [Ni(mnt)(2)](-), along with the quadrupole tensor for nitrogen. These measurements give pi electron spin densities of rho(C1) approximately 0.03 in the C1 2p(z)() orbital, rho(C2) < 0.