Proteins from Avastin® (bevacizumab) Show Tyrosine Nitrations for which the Consequences Are Completely Unclear

Avastin® (bevacizumab) is a protein drug widely used for cancer treatment although its further use is questionable due to serious side effects reported. As no systematic proteomic study on posttranslational modifications (PTMs) was reported so far, it was the aim of the current study to use a gel-based proteomics method for determination of Avastin®-protein(s)

Oxidative protein labeling in mass-spectrometry-based proteomics

Oxidation of proteins and peptides is a common phenomenon, and can be employed as a labeling technique for mass-spectrometry-based proteomics. Nonspecific oxidative labeling methods can modify almost any amino acid residue in a protein or only surface-exposed regions. Specific agents may label reactive functional groups in amino acids, primarily cysteine, methionine, tyrosine, and tryptophan. Nonspecific radical intermediates (reactive oxygen, nitrogen, or halogen species) can be produced by chemical, photochemical, electrochemical, or enzymatic methods. More targeted oxidation can be achieved by chemical reagents but also by direct electrochemical oxidation, which opens the way to instrumental labeling methods. Oxidative labeling of amino acids in the context of liquid chromatography(LC)–mass spectrometry (MS) based proteomics allows for differential LC separation, improved MS ionization, and label-specific fragmentation and detection. Oxidation of proteins can create new reactive groups which are useful for secondary, more conventional derivatization reactions with, e.g., fluorescent labels. This review summarizes reactions of oxidizing agents with peptides and proteins, the corresponding methodologies and instrumentation, and the major, innovative applications of oxidative protein labeling described in selected literature from the last decade

Exacerbative role of vitamin A on radiation damage in vivo

Purpose In our study, after applying a single dose of 612 cGy irradiation, we aimed to observe the role of free radicals on tissue damage in liver caused by irradiation, by measuring the nitric oxide (NO) level, superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzyme activities, and the amount of thiobarbituric acid reactive substance (TBARS), which is an indicator of free-radical damage. On the other hand we investigated whether tissue damage can be prevented by vitamin A or not. Materials and methods The study was performed on three groups: (1) the control group, (2) the group to which irradiation was administrated, and (3) the group which was given irradiation + vitamin A. The irradiation group of animals was given a single dose of gamma irradiation at a sublethal dose. In the group to which both irradiation and vitamin A were administered, vitamin A was given for 2 days prior to irradiation. The amount of NO was measured by electron spin resonance (ESR) spectroscopy, whereas SOD, GPx and TBARS were measured by spectrophotometry. Results and conclusion As a result of irradiation-mediated tissue damage in liver, we observed a NO loss and an increase in TBARS amount. Administration of vitamin A before irradiation resulted in an increase in both NO and TBARS and a decrease in SOD and GPx enzyme activities. Together, these data indicate that vitamin A may play an exacerbative role in free-radical-mediated tissue damage

Site-Specific Incorporation of 3-Nitrotyrosine as a Probe of pK[subscript a] Perturbation of Redox-Active Tyrosines in Ribonucleotide Reductase

E. coli ribonucleotide reductase catalyzes the reduction of nucleoside 5′-diphosphates into 2′-deoxynucleotides and is composed of two subunits: α2 and β2. During turnover, a stable tyrosyl radical (Y•) at Y[subscript 122-]β2 reversibly oxidizes C[subscript 439] in the active site of α2. This radical propagation step is proposed to occur over 35 Å, to use specific redox-active tyrosines (Y[subscript 122] and Y[subscript 356] in β2, Y[subscript 731] and Y[subscript 730] in α2), and to involve proton-coupled electron transfer (PCET). 3-Nitrotyrosine (NO[subscript 2]Y, pK[subscript a] 7.1) has been incorporated in place of Y[subscript 122], Y[subscript 731], and Y[subscript 730] to probe how the protein environment perturbs each pK[subscript a] in the presence of the second subunit, substrate (S), and allosteric effector (E). The activity of each mutant is 9.6. X-ray crystal structures have been obtained for all [NO[subscript 2]Y]-α2 mutants (2.1−3.1 Å resolution), which show minimal structural perturbation compared to wt-α2. Together with the pK[subscript a] of the previously reported NO[subscript 2]Y[subscript 356-]β2 (7.5 in the α2/S/E complex; Yee, C. et al. Biochemistry 2003, 42, 14541−14552), these studies provide a picture of the protein environment of the ground state at each Y in the PCET pathway, and are the starting point for understanding differences in PCET mechanisms at each residue in the pathway.National Institutes of Health (U.S.) (GM29595