title: Ovalbumin labeling with p-hydroxymercurybenzoate: The effect of different denaturing agents and the kinetics of reaction.
abstract: 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). In addition to chemical denaturation, thermal denaturation was also tested. The protein was pre-column simultaneously denatured and derivatized, and the pHMB-labeled denatured OVA complexes were analyzed by size exclusion chromatography (SEC) coupled online with chemical vapor generation-atomic fluorescence spectrometry (CVG-AFS). The number of -SH groups titrated greatly depends on the protein structure in solution. Indeed, we found that, depending on the adopted denaturing conditions, OVA gave different aggregate species that influence the complexation process. The results were compared with those obtained by a common alternative procedure for the titration of -SH groups that employs monobromobimane (mBBr) as tagging molecule and molecular fluorescence spectroscopy as detection technique. We also investigated the labeling kinetics for denatured OVA and pHMB, finding that the 4 thiolic groups of OVA have a very different reactivity toward mercury labeling, in agreement with previous studies.
authors.affiliation: Istituto di Chimica dei Composti Organo Metallici (ICCOM) - National Research Council of Italy (CNR), UOS di Pisa, 56124 Pisa, Italy; Dipartimento di Chimica e Chimica Industriale, Università di Pisa, 56124 Pisa, Italy.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Campanella
authors.affiliation: Istituto di Chimica dei Composti Organo Metallici (ICCOM) - National Research Council of Italy (CNR), UOS di Pisa, 56124 Pisa, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Onor
authors.affiliation: Dipartimento di Chimica e Chimica Industriale, Università di Pisa, 56124 Pisa, Italy.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Biancalana
authors.affiliation: Istituto di Chimica dei Composti Organo Metallici (ICCOM) - National Research Council of Italy (CNR), UOS di Pisa, 56124 Pisa, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: D'Ulivo
authors.affiliation: Istituto di Chimica dei Composti Organo Metallici (ICCOM) - National Research Council of Italy (CNR), UOS di Pisa, 56124 Pisa, Italy. Electronic address: bramanti@pi.iccom.cnr.it.
authors.forename: Emilia
authors.initials: E
authors.lastname: Bramanti
mesh_terms: D000818:Animals; D002645:Chickens; D002850:Chromatography, Gel; D006902:Hydroxymercuribenzoates / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008628:Mercury / Q000737:chemistry / Q000378:metabolism*; D010047:Ovalbumin / Q000378:metabolism*; D011489:Protein Denaturation; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013194:Staining and Labeling
chemical_list: D006902:Hydroxymercuribenzoates; C030601:4-hydroxymercuribenzoate; D012967:Sodium Dodecyl Sulfate; D010047:Ovalbumin; D008628:Mercury
keywords: Atomic fluorescence spectrometry; Denaturation; Hyphenated techniques; Thiolic proteins; p-Hydroxymercurybenzoate