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Ovalbumin labeling with p-hydroxymercurybenzoate: The effect of different denaturing agents and the kinetics of reaction.
Anal Biochem
August 2015
Istituto di Chimica dei Composti Organo Metallici (ICCOM) - National Research Council of Italy (CNR), UOS di Pisa, 56124 Pisa, Italy. Electronic address:
The aim of our study was to investigate how denaturing agents commonly used in protein analysis influence the labeling between a reactive molecule and proteins. For this reason, we investigated the labeling of ovalbumin (OVA) as a globular model protein with p-hydroxymercurybenzoate (pHMB) in its native state (phosphate buffer solution) and in different denaturing conditions (8 molL(-1) urea, 3 molL(-1) guanidinium thiocyanate, 6 molL(-1) guanidinium chloride, 0.2% sodium dodecyl sulfate, and 20% methanol).
View Article and Find Full Text PDFMethemoglobin reduction by NADH-cytochrome b(5) reductase in Propsilocerus akamusi larvae.
Comp Biochem Physiol B Biochem Mol Biol
July 2015
Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, Tokyo 101-8308, Japan. Electronic address:
For oxygen respiration, a methemoglobin (metHb) reduction system is needed in the cell because metHb cannot bind oxygen. We examined the insect Propsilocerus akamusi larvae to elucidate the metHb reduction system in an organism that inhabits an oxygen-deficient environment. NADH-dependent reduction of metHb in coelomic fluid suggested the coexistence of cytochrome b5 reductase (b5R) and cytochrome b5 with hemoglobin in the fluid and that these proteins were involved in physiological metHb reduction in the larvae.
View Article and Find Full Text PDFDetermination of sub-nanomolar levels of low molecular mass thiols in natural waters by liquid chromatography tandem mass spectrometry after derivatization with p-(hydroxymercuri) benzoate and online preconcentration.
Anal Chem
January 2015
Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden.
Low molecular mass (LMM) thiols is a diverse group of compounds, which play several important roles in aquatic ecosystems, even though they typically occur at low concentrations. Comprehensive studies of LMM thiols in natural waters have so far been hampered by selectivity and limit of detection constraints of previous analytical methods. Here, we describe a selective and robust method for the quantification of 16 LMM thiols in natural waters.
View Article and Find Full Text PDFDirect, simple derivatization of disulfide bonds in proteins with organic mercury in alkaline medium without any chemical pre-reducing agents.
Anal Chim Acta
September 2014
National Research Council of Italy, C.N.R., Istituto di Chimica dei Composti Organo Metallici-ICCOM- UOS Pisa, Area di Ricerca, Via G. Moruzzi 1, 56124 Pisa, Italy. Electronic address:
In this work we have studied the derivatization of protein disulfide bonds with p-Hydroxymercurybenzoate (pHMB) in strong alkaline medium without any preliminary reduction. The reaction has been followed by the determination of the protein-pHMB complex using size exclusion chromatography coupled to a microwave/UV mercury oxidation system for the on-line oxidation of free and protein-complexed pHMB and atomic fluorescence spectrometry (SEC-CVG-AFS) detection. The reaction has been optimized by an experimental design using lysozyme as a model protein and applied to several thiolic proteins.
View Article and Find Full Text PDFMicrowave photochemical reactor for the online oxidative decomposition of p-hydroxymercurybenzoate (pHMB)-tagged proteins and their determination by cold vapor generation-atomic fluorescence detection.
Anal Chem
December 2013
National Research Council of Italy, C.N.R. , Istituto di Chimica dei Composti Organo Metallici - ICCOM - UOS Pisa, Area di Ricerca, Via G. Moruzzi 1, 56124 Pisa, Italy.
A novel method is presented for the characterization and determination of thiolic proteins. After the labeling with p-hydroxymercurybenzoate, the pHMB-labeled proteins underwent on-line oxidation with a novel microwave (MW)/UV photochemical reactor, followed by cold vapor generation-atomic fluorescence spectrometry (CVG-AFS) detection. The MW/UV process led to the conversion of pHMB to Hg(II) with a yield of 89.
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