A Comparison between the Sulfhydryl Reductants Tris(2-carboxyethyl)phosphine and Dithiothreitol for Use in Protein Biochemistry

Getz, Elise Burmeister; Xiao, Ming; Chakrabarty, Tania; Cooke, Roger; Selvin, Paul R.

doi:10.1006/abio.1999.4203

Cited by 430 publications

(322 citation statements)

References 23 publications

(27 reference statements)

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“…Finally, a sample with 1 mM TCEP present during AMS modification contained a maximum of only 5 AMS units. This result is consistent with previous reports showing that the efficiency of maleimide labeling is significantly decreased by the presence of reductants (35). If SDS is not included in the modification reaction, only three of seven cysteines are modified regardless of the reductant and conditions used, implying that the other four cysteines are not easily accessible.…”

Section: Resultssupporting

confidence: 92%

Yeast Cox17 Solution Structure and Copper(I) Binding

Abajian

Yatsunyk

Ramirez

et al. 2004

Journal of Biological Chemistry

101

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Section: Resultssupporting

confidence: 92%

Yeast Cox17 Solution Structure and Copper(I) Binding

Abajian

Yatsunyk

Ramirez

et al. 2004

Journal of Biological Chemistry

101

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“…Importantly, CPM is not fluorescent unless conjugated to thiols, a feature that further prevents undesired non-specific noise. In the reducing step, DTT was replaced by TCEP, a reagent that does not interfere with the reaction between maleimide and thiols [30]. Therefore, washing steps in which the samples would be exposed to atmospheric oxygen in the absence of reductants can be omitted.…”

Section: Discussionmentioning

confidence: 99%

A FRET-based method to study protein thiol oxidation in histological preparations

Mastroberardino

Orr

et al. 2008

Free Radical Biology and Medicine

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Cysteine residues in proteins have important biological roles. For example, disulfides bonds are important structural elements; additionally, reversible oxidation of thiols to disulfides functions as a molecular switch and constitutes an early response to oxidative damage Because organs are heterogeneous structures composed of diverse cell types, there is a compelling need for a histological approach to investigate thiol oxidation in situ in order to address the role of specific cell types in oxidative imbalance. Here we describe a fluorescence technique -which can be used in association with standard immunological staining procedures -to detect variations in disulfides in histological preparations. Moreover, by monitoring the fluorescence resonance energy transfer (FRET) between a labeled specific primary antibody and the thiol probe described here, this method can detect thiol oxidation in candidate proteins of interest. When applied to an animal model of Parkinson's disease, our technique demonstrated that thiol oxidation occurs selectively in the dopaminergic neurons of the substantia nigra, the same neurons that are lost selectively in the disease. In summary, this technique provides a new, powerful tool to provide further understanding of oxidative imbalance, a phenomenon common to many diseases. Keywords fluorescence microscopy; FRET; oxidative stress; cysteine; thiols; disulfides; Parkinson's disease Thiols are the functional groups in the side chain of the amino acid, cysteine. Depending upon the oxido-reductive (redox) status of the surrounding environment, thiols exist either in the reduced form of free thiols (-SH) or oxidized to disulfides (-S-S-). Disulfides are well known to play an important structural role, contributing to the maintenance of the proper folding in proteins. More recently it was found that the formation of disulfides in proteins also exerts critical regulatory functions: vital mechanisms such as protein import, regulation of signal transduction cascades, regulation of the activity of transcription factors and proper function of the mitochondrial electron transport system rely on disulfide oxidation or reduction [1][2][3][4]. Moreover, the formation of disulfides represents an early, reversible response to oxidative *Corresponding author: mastroberardinopg@upmc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Increasing interest in the study of thiols has led to the development of a variety of technical approaches to detect thiols and disulfides. In particular, several spectrophotometry-based protocols were developed to measure t...

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“…When DTT and TCEP were used, their concentrations were Ն1 mM, for which there is on average Ն1 molecule of reducing agent/10 nm 3 of solution. Trialkylphosphines such as TCEP are highly specific for disulfides and reduce S-S more rapidly than does DTT (16). In addition to being water-soluble, TCEP is also highly stable in basic solutions (17), unlike DTT, which readily oxidizes above pH 7.5.…”

Section: Forced Unfolding Of Vcammentioning

confidence: 99%

Chemistry on a Single Protein, Vascular Cell Adhesion Molecule-1, during Forced Unfolding

Bhasin

Carl

Harper

et al. 2004

Journal of Biological Chemistry

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Proteins of many types experience tensile forces in their normal function, and vascular cell adhesion molecule-1 (VCAM-1) is typical in this. VCAM has seven Ig domains, and each has a disulfide bond (-S-S-) buried in its core that covalently stabilizes about half of each domain against unfolding. VCAM is extended here by single molecule atomic force microscopy in the presence or absence of reducing agents. In the absence of reducing agent, a sawtooth pattern of forced unfolding reveals an average period and total length consistent with disulfide locations in VCAM. With increasing reducing agent, accessible disulfides are specifically reduced (to SH); the average period for unfolding increases up to saturation together with additional metrics of unfolding. Steered molecular dynamics simulations of unfolding indeed show that the core disulfide bond is solventexposed in the very earliest stages of protein extension. Michaelis-Menten kinetics emerge with reduction catalyzed by force ( reduction ϳ 10 ؊4 s). The results establish single molecule reduction, one bond at a time, and show that mechanical forces can play a key role in modulating the redox state of cell adhesion proteins that are invariably stressed in cell adhesion.

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A Comparison between the Sulfhydryl Reductants Tris(2-carboxyethyl)phosphine and Dithiothreitol for Use in Protein Biochemistry

Cited by 430 publications

References 23 publications

Yeast Cox17 Solution Structure and Copper(I) Binding

Yeast Cox17 Solution Structure and Copper(I) Binding

A FRET-based method to study protein thiol oxidation in histological preparations

Chemistry on a Single Protein, Vascular Cell Adhesion Molecule-1, during Forced Unfolding

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