An Amplified Assay for Thiols Based on Reactivation of Papain

Singh, Rajeeva; Blättler, Walter A.; Collinson, Albert R.

doi:10.1006/abio.1993.1384

Cited by 60 publications

(62 citation statements)

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“…A kinetic, spectrophotometric amidase assay with N α -benzoyl-L-arginine-4-nitroanilide hydrochloride (L-BAPNA, B3279, Sigma) as substrate was used to measure papain activity. 73,74 Assays contained papain (2-5 μM), 50 mM Tris pH 7.1, 1 mM EDTA and were initiated by the addition of L-BAPNA (2 mM). The absorbance increase at 410 nm was recorded on a Uvikon XS spectrophotometer (Research Instruments International).…”

Section: Experimental Preparation Enzymatic Assay and Chemical Modifmentioning

confidence: 99%

A chemical approach for detecting sulfenic acid-modified proteins in living cells

et al. 2008

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Oxidation of the thiol functional group in cysteine (Cys-SH) to sulfenic (Cys-SOH), sulfinic (Cys-SO 2 H) and sulfonic acids (Cys-SO 3 H) is emerging as an important post-translational modification that can activate or deactivate the function of many proteins. Changes in thiol oxidation state have been implicated in a wide variety of cellular processes and correlate with disease states but are difficult to monitor in a physiological setting because of a lack of experimental tools. Here, we describe a method that enables live cell labeling of sulfenic acidmodified proteins. For this approach, we have synthesized the probe DAz-1, which is chemically selective for sulfenic acids and cell permeable. In addition, DAz-1 contains an azide chemical handle that can be selectively detected with phosphine reagents via the Staudinger ligation for identification, enrichment and visualization of modified proteins. Through a combination of biochemical, mass spectrometry and immunoblot approaches we characterize the reactivity of DAz-1 and highlight its utility for detecting protein sulfenic acids directly in mammalian cells. This novel method to isolate and identify sulfenic acid-modified proteins should be of widespread utility for elucidating signaling pathways and regulatory mechanisms that involve oxidation of cysteine residues.

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Section: Experimental Preparation Enzymatic Assay and Chemical Modifmentioning

confidence: 99%

A chemical approach for detecting sulfenic acid-modified proteins in living cells

et al. 2008

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“…3). In addition, higher than optimal calcium (19), including thioredoxin (20), are able to restore activity following oxidative inactivation. A similar lack of thiol specificity differentiates thiocalsin from the kallikrein serine proteases (21).…”

Section: (-Sh Hs-) (--) (-S-s-)mentioning

confidence: 99%

Thiocalsin: a thioredoxin-linked, substrate-specific protease dependent on calcium.

Besse

Wong

Kobrehel

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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“…Hydrogen sulfide concentration was measured using the enzymatic assay of Singh et al (1993) as modified by Freytag et al (2001). This assay measures the concentration of all sulfide species, though at bottom-water pH (7.7, Aharon & Fu 2000) the majority (86%) of total sulfide is in the form of HS -.…”

Section: Methodsmentioning

confidence: 99%

Succession of hydrocarbon seep communities associated with the long-lived foundation species Lamellibrachia luymesi

Cordes¹,

Hourdez²,

Predmore³

et al. 2005

Mar. Ecol. Prog. Ser.

130

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The vestimentiferan tubeworm Lamellibrachia luymesi lives for over 250 yr and forms aggregations of hundreds to thousands of individuals at hydrocarbon seep sites in the Gulf of Mexico. A succession model has been proposed where changes in the abiotic environment within tubeworm aggregations lead to shifts in the associated community structure. Here, we test this model using new data from communities associated with 13 tubeworm aggregations. We quantify the temporal scale of succession by using a population-growth model to estimate aggregation age. In older tubeworm aggregations, biomass, density, and number of species m -2 tube surface area decline. In addition, primary producer, primary consumer, and endemic species comprise a smaller proportion of the tubeworm-associated community in old aggregations. These changes in the associated community are attributed to lower epibenthic sulfide concentrations in older aggregations. A number of species' densities show strong correlations to sulfide concentration, and similarity between communities is best explained by a combination of aggregation age and sulfide concentration. Together, these data demonstrate the role of hydrogen sulfide in this seep habitat and the effect that the hypothesized reduction of epibenthic sulfide concentration by L. luymesi would have on the associated community.

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An Amplified Assay for Thiols Based on Reactivation of Papain

Cited by 60 publications

References 0 publications

A chemical approach for detecting sulfenic acid-modified proteins in living cells

A chemical approach for detecting sulfenic acid-modified proteins in living cells

Thiocalsin: a thioredoxin-linked, substrate-specific protease dependent on calcium.

Succession of hydrocarbon seep communities associated with the long-lived foundation species Lamellibrachia luymesi

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