Delineating Heme-Mediated versus Direct Protein Oxidation in Peroxidase-Activated Cytochrome c by Top-Down Mass Spectrometry.

Oxidation of key residues in cytochrome c (cyt c) by chloramine T (CT) converts the protein from an electron transporter to a peroxidase. This peroxidase-activated state represents an important model system for exploring the early steps of apoptosis. CT-induced transformations include oxidation of the distal heme ligand Met80 (MetO, +16 Da) and carbonylation (LysCHO, -1 Da) in the range of Lys53/55/72/73. Remarkably, the 15 remaining Lys residues in cyt c are not susceptible to carbonylation. The cause of this unusual selectivity is unknown. Here we applied top-down mass spectrometry (MS) to examine whether CT-induced oxidation is catalyzed by heme. To this end, we compared the behavior of cyt c with (holo-cyt c) and without heme (apoSS-cyt c). CT caused MetO formation at Met80 for both holo- and apoSS-cyt c, implying that this transformation can proceed independently of heme. The aldehyde-specific label Girard's reagent T (GRT) reacted with oxidized holo-cyt c, consistent with the presence of several LysCHO. In contrast, oxidized apo-cyt c did not react with GRT, revealing that LysCHO forms only in the presence of heme. The heme dependence of LysCHO formation was further confirmed using microperoxidase-11 (MP11). CT exposure of apoSS-cyt c in the presence of MP11 caused extensive nonselective LysCHO formation. Our results imply that the selectivity of LysCHO formation at Lys53/55/72/73 in holo-cyt c is caused by the spatial proximity of these sites to the reactive (distal) heme face. Overall, this work highlights the utility of top-down MS for unravelling complex oxidative modifications.

A one-step synthesis of rare-earth phosphate-borosilicate glass composites.

A new 1-step method for synthesizing glass-ceramic composites consisting of rare earth phosphates (REPO4) dispersed in borosilicate glass (BG) is reported herein as an alternative to the 2-step approach that is traditionally used. The effect of annealing time and annealing temperature on the formation of the 1-step glass-ceramic composites was investigated. Backscattered electron images and energy dispersive X-ray maps were collected to observe the morphology and chemical distribution in the glass-ceramic composites. X-ray diffraction was used to study the long-range order and X-ray absorption near edge spectroscopy was used to study the local environment of La, Y, P and Si. All analyses showed glass-ceramic composites made by the 1 and 2-step methods were similar to each other except for the Si L2,3-edge XANES spectra, which showed a slight change between the glass-ceramic composite materials made by the different synthesis methods. Xenotime-type phosphates (YPO4) were observed to be more soluble in the borosilicate glass than the monazite-type phosphates (LaPO4). This was attributed to the difference in the field strength of the rare-earth ions as a result of the difference in the ionic radii. Glass-ceramic composites made by the 1-step method were shown to form in 1 day at 1100 °C and in 3 days at 1000 °C without a significant change in glass or ceramic composition compared to the 1-step composite synthesized at 1100 °C for 3 days.