cytochrome-c-t and tris(2-carboxyethyl)phosphine

Airborne particulate matter (PM) contains several quinones, which are able to generate reactive oxygen species impacting on cell viability. A method able to detect and quantify PM oxidative potential, based on the cytochrome c (cyt-c) reduction by means of superoxide anion produced through quinones redox cycling in the presence of reducing agents, is here described. Tris(2-carboxyethyl)phosphine resulted to be the most efficient reducing agent among the ones tested. The procedure included rapid particles extraction, followed by two alternative analytical methods, a spectrophotometric assay based on the initial rate of cyt-c reduction at 550 nm, and an amperometric assay, based on self-assembled monolayers modified gold electrodes. The smallest amount of PM needed to obtain an evaluable signal is 2 μg. The described procedure may represent a starting point to develop devices for PM measurements in polluted atmospheric environments.

Topics: Cytochromes c; Electrodes; Environmental Monitoring; Gold; Oxidation-Reduction; Particulate Matter; Phosphines; Quinones; Reactive Oxygen Species; Superoxides

In this work, the reactivity of the citostatic drugs such as oxaliplatin, cisplatin and carboplatin towards proteins and the stability of Pt-protein complexes along their storage were evaluated. Neither native-PAGE nor nrSDS-PAGE seems to be suitable for the separation of carboplatin-binding proteins. A reducing electrophoretic separation procedure able to maintain the integrity of oxaliplatin-protein complexes has been developed. The method is based on SDS-PAGE under conditions provided by the thiol-free reducing agent tris (2-carboxyethyl) phosphine (TCEP), which allowed the separation of oxaliplatin-binding proteins in narrow bands with almost quantitative recoveries. Different amounts of platinum-bound protein bands covering the range 0.3-2.0 μg were excised and mineralised for platinum determination, showing good linearity. Limits of detection for a mixture of five standard proteins (transferrin, albumin, carbonic anhydrase, myoglobin and cytochrome c) incubated with oxaliplatin were within the range 11.0-44.0 pg of platinum, which were satisfactory for their application to biological samples. The suitability of the TCEP-based SDS-PAGE for the separation of platinum-enriched protein fractions of a kidney cytosol from a rat treated with oxaliplatin was demonstrated. The identification of high Pt to protein ratio cytosolic fractions was carried out by separating the cytosolic platinum-binding proteins by SEC-ICP-MS. Several cytosolic renal proteins were identified in those gel bands containing platinum-enriched protein fractions using nLC-ESI-LTQ-MS/MS after in-gel digested with trypsin. In addition, fractions containing platinum-enriched proteins with lower theorical molecular weight were directly analysed by nLC-ESI-LTQ-MS/MS after in-solution tryptic digestion allowing protein identification.

Topics: Animals; Carbonic Anhydrases; Carboplatin; Cisplatin; Cytochromes c; Electrophoresis, Polyacrylamide Gel; Kidney; Limit of Detection; Myoglobin; Organoplatinum Compounds; Oxaliplatin; Peptide Fragments; Phosphines; Platinum; Protein Binding; Proteolysis; Rats; Serum Albumin; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tandem Mass Spectrometry; Transferrin; Trypsin

We studied the electrophoretic behavior of basic proteins (cytochrome c and histone III) and developed a carbamylation method that normalizes their electrophoretic size separation and improves the accuracy of their relative molecular mass determined electrophoretically. In capillary zone electrophoresis with cationic hitchhiking, native cytochrome c does not sufficiently bind cationic surfactants due to electrostatic repulsion between the basic protein and cationic surfactant. Carbamylation suppresses the strong positive charge of the basic proteins and results in more accurate relative molecular masses.

Topics: Animals; Biochemical Phenomena; Cations; Cattle; Cytochromes c; Electrophoresis, Capillary; Hydrophobic and Hydrophilic Interactions; Molecular Weight; Phosphines; Proteins

Cross-links between amino acid residues in close proximity can provide distance constraints for the validation of models of the 3D structure proteins. The mapping of cross-links by the identification of linked peptides in proteolytic digests is facilitated by cleavable cross-linkers that enable isolation of the cleavage products while preserving information about the linkage. We present an amine-specific cross-linker, bis(succinimidyl)-3-azidomethyl glutarate (BAMG), that fulfils these requirements. Two parallel reaction pathways are induced by tris(carboxyethyl)phosphine (TCEP) in cross-linked peptides from BAMG-treated cytochrome c. One pathway leads to cleavage of the cross-linked species, while in the other the azido group of BAMG is reduced to an amino group without cleavage. Cross-linked peptides and peptides modified by partially hydrolysed BAMG yield distinct sets of TCEP-induced reaction products. These can be isolated by reversed-phase diagonal chromatography and identified by mass spectrometry to reveal the identity of the parent compounds. The ease with which cross-link-derived reaction products can be isolated and identified indicates that the mapping of cross-links in complex biological assemblies and mixtures of protein complexes might become feasible in the near future.

Topics: Animals; Azides; Chromatography, High Pressure Liquid; Cross-Linking Reagents; Cytochromes c; Horses; Lysine; Molecular Sequence Data; Molecular Structure; Peptides; Phosphines; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization