Measurement of Protein Sulfenic Acid Content

Abstract

Protein sulfenic acids are reactive, reversibly oxidized cysteinyl residues with roles in redox catalysis and regulation. Detection and quantification of these species in proteins is accomplished through chemical modification by reagents such as 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD chloride), 2-nitro-5-thiobenzoate (TNB), dimedone, or derivatives of dimedone, followed by UV-visible spectroscopy or mass spectrometric analysis.

NemR Is a Bleach-sensing Transcription Factor

“…The simplest hypothesis to explain this result was that, in the absence of other cysteines, Cys-106 might undergo reversible sulfenic acid formation, the immediate consequence of RCS-mediated chlorination of thiol groups (4). Both reduced thiols and sulfenic acids can be detected using the probe NBD-Cl, which forms adducts with absorption maxima of 420 nm for thiols and 350 nm for sulfenic acid (31). We recorded the UV spectrum of N-chlorotaurine-oxidized NemR 5C-S(C106) in the presence of NBD-Cl but were unable to detect any significant sulfenic acid adducts in our sample (data not shown).…”

“…5Ј-CTA AAC GGC AGG CGT CGC AAT AAT GTT TTT CAT ATG AAT ATC CTC CTT AGT TCC-3Ј [3] 5Ј -CCT GCC GTT TAG CAG GCA TTT TTT ATC ACC AGA CGA CCG GGA GCC TTT ATG GCG GTG TAG GCT GGA GCT GCT TC-3Ј [4] 5Ј-AAT GTT GTT TTA GTA TAG CGG CGG CTT TAC GCC GCT CGC AAT GTT GGA TTA CAT ATG AAT ATC CTC CTT AG-3Ј [5] 5Ј-ATA CTA AAA CAA CAT TTT GAA TCT GTT AGC CAT TTT GAG GAT AAA AAG ATG GTG TAG GCT GGA GCT GCT TC-3Ј [6] 5Ј-TCG ATG CAG TAA AGA TGC GGG CGC GAT GAG TTC ACG CCC GGC AGG AGA TTA CAT ATG AAT ATC CTC CTT AGG-3Ј [7] 5Ј-ATG CAT ATG AAC AAA CAC ACC GAA CAT G-3Ј [8] 5Ј-CTT aag ctt act acc gcg tgg cac cag AAC GGC AGG CGT CGC AAT AAT-3Ј [9] 5Ј-TTC GAA TTC TGG TCT ACT ACA CTC CAA CGC A-3Ј [10] 5Ј-CTT aag ctt CTA AAC GGC AGG CGT CGC AAT AAT-3Ј [11] 5Ј-CAT CAG TGG TTC TCT GAC AGT AA-3Ј [12] 5Ј-TTA CTG TCA GAG AAC CAC TGA TG-3Ј [13] 5Ј-CAA ACA CTG AAC CAG TTT AGC CAA CAT GGA ACC ATC-3Ј [14] 5Ј-GAT GGT TCC ATG TTG GCT AAA CTG GTT CAG TGT TTG-3Ј [15] 5Ј-CTC TGC CGA AGT GAG CGA TCT GTC AG-3Ј [16] 5Ј-CTG ACA GAT CGC TCA CTT CGG CAG AG-3Ј [17] 5Ј-GAA AAT GGC CGT GAG AAC CAT AGC TTA ACC TTT TGT GGC GAA CC-3Ј [18] 5Ј-GAA CCA TTG TTT AAC CTT TAG CGG CGA ACC GCT GCA ACA G-3Ј [19] 5Ј-GGC GAG CAA CTT AGC CTG CAA CGT G-3Ј [20] 5Ј-TAG CAA CTT TGC TTG CAC TAG ACC GAC TGG TCT ACT ACA CTC CAA CGC ATG AAC AAA CAC ACC GAA CAT G-3Ј [21] 5Ј-GAA GCA GCT CCA GCC TAC ACC TAA ACG GCA GGC GTC GCA ATA AT-3Ј [22] 5Ј-ATT ATT GCG ACG CCT GCC GTT TAG GTG TAG GCT GGA GCT GCT TC-3Ј [23] 5Ј-TTT TTC AGA TGA CAT AAA GGC TCC CGG TCG TCT GGT GAT AAA AAA TGC CTG CAT ATG AAT ATC CTC CTT AG-3Ј [24] 5Ј-AAG AAC TTA CCT GGT CTT GAC ATC-3Ј [25] 5Ј-CAG TTT ATC ACT GGC AGT CTC CTT-3Ј [26] 5Ј-GGT CCT TCT ATC ACT ACT TTC GCT CT-3Ј [27] 5Ј-GTT TTA CTG TCA GGC AAC CAC TGA T-3Ј [28] 5Ј-AGG TGC ATA CAC AGT AGA AAA AGC TG-3Ј [29] 5Ј-TAC AAC GTC GGG TAA TCG GTA TAG-3Ј [30] 5Ј-TAA ATA CGA ACT CGG CAC TGC TTA T-3Ј [31] 5Ј-GCG TCT TTC TCT TCG ATT AAC TCA-3Ј …”

Chemical approaches to detect and analyze protein sulfenic acids

“…An important approach to quantify sulfenic acids in proteins relies on their propensity to generate disulfide bonds with thiol groups. In particular, the bright yellow, small thiolcontaining molecule TNB, described above as the product of DTNB reduction, reacts stoichiometrically with sulfenic acids and is well suited for this purpose (Poole & Claiborne, 1989;Ellis & Poole, 1997a;Poole & Ellis, 2002;Poole, 2008;Rehder & Borges, 2010b). As long as the TNB solution is freshly prepared, is protected as much as possible from air oxidation, and does not include any residual DTNB or DTT, the loss of the strong 412 nm absorbance of TNB upon addition of a sulfenic acid-containing sample (if the sulfenic acid is sufficiently exposed) correlates directly with the sulfenic acid content.…”

“…The experimental approaches can be tailored for the analysis of purified proteins, mixtures of proteins, or even cellular samples, and both targeted and global analyses of oxidation-sensitive CySOH can be conducted, as described briefly below and summarized in Figure 9. For further methodological details and advice, the reader may wish to consult additional literature on the subject (Poole, 2008;Poole & Nelson, 2008;Klomsiri et al, 2010;Nelson et al, 2010;.…”

Nontypeable Haemophilus influenzae redox recycling of protein thiols promotes resistance to oxidative killing and bacterial survival in biofilms in a smoke related infection model

“…To further investigate the susceptibility and redox state of the isogenic mutants, we purified oxidized bacterial proteins based on affinity of cysteine sulfenic acid (CSA) for the nucleophile 1,3cyclopentanedione (BP1) (45)(46)(47). Biotin-linked BP1 was used to purify CSA-modified proteins from bacterial lysates, which were quantified by SDS/PAGE and silver stain.…”