Selective reduction of disulfides by tris(2-carboxyethyl)phosphine

Burns, John A.; Butler, James C.; Moran, John T.; Whitesides, George M.

doi:10.1021/jo00008a014

Cited by 650 publications

(584 citation statements)

References 2 publications

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“…Ruegg and Rudinger (1977) briefly discussed TCEP, comparing it unfavorably to tributylphosphine in terms of reactivity with proteins. Report of a convenient synthesis of TCEP, and its selective behavior toward simple organic disulfides (Burns et al, 1991), was the stimulus for the present work. Buckwalter et al (1992) independently described the use of TCEP for selective reduction of a disulfide in somatotropin.…”

Section: Reduction Of Disulfides By Trialkylphosphinesmentioning

confidence: 99%

“…In its action on organic disulfides, TCEP was found to be kinetically selective in that rate of reduction was dependent on the degree of strain in the disulfide rather than on its thermodynamic stability (Burns et al, 1991). An obvious possibility for peptide analysis, therefore, would be to carry out the reduction in a stepwise fashion: stoichiometrically reduce one bridge, alkylate it, and then reduce another, etc.…”

Section: Selectivity and Kinetics Of Reductionmentioning

confidence: 99%

“…Trialkylphosphines, on the other hand, are highly reactive toward disulfides and can be employed at low pH (Ruegg & Rudinger, 1977). The water-soluble (Levison et al, 1969;Burns et al, 1991) has proved to be excellent for this purpose. Unlike many other phosphines, it is a stable crystalline compound applied readily in aqueous medium.…”

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confidence: 99%

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Disulfide structures of highly bridged peptides: A new strategy for analysis

1993

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A new approach is described for analyzing disulfide linkage patterns in peptides containing tightly clustered cystines. Such peptides are very difficult to analyze with traditional strategies, which require that the peptide Chain be split between close or adjacent Cys residues. The water-soluble tris-(2-~arboxyethyl)-phosphine (TCEP) reduced disulfides at pH 3, and partially reduced peptides were purified by high performance liquid chromatography with minimal thiol-disulfide exchange. Alkylation of free thiols, followed by sequencer analysis, provided explicit assignment of disulfides that had been reduced. Thiol-disulfide exchange occurred during alkylation of some peptides, but correct deductions were still possible. Alkylation competed best with exchange when peptide solution was added with rapid mixing to 2.2 M iodoacetamide. Variants were developed in which up to three alkylating agents were used to label different pairs of thiols, allowing a full assignment in one sequencer analysis. Model peptides used included insulin (three bridges, intra-and interchain disulfides; -Cys.Cys-pair), endothelin and apamin (two disulfides; -Cys.x.Cys-pair), conotoxin GI and isomers (two disulfides; -Cys.Cys-pair), and bacterial enterotoxin (three bridges within 13 residues; two -Cys.Cys-pairs). With insulin, all intermediates in the reduction pathway were identified; with conotoxin GI, analysis was carried out successfully for all three disulfide isomers. In addition to these known structures, the method has been applied successfully to the analysis of several previously unsolved structures of similar complexity. Rates of reduction of disulfide bonds varied widely, but most peptides did not show a strongly preferred route for reduction.

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Section: Reduction Of Disulfides By Trialkylphosphinesmentioning

confidence: 99%

Section: Selectivity and Kinetics Of Reductionmentioning

confidence: 99%

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Disulfide structures of highly bridged peptides: A new strategy for analysis

1993

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“…The cysteinecontaining peptide (2.5 mg in bidistilled water) was reduced with an equimolar amount of Tris-(2-carboxyethyl)-phosphine hydrochloride (AppliChem, Darmstadt, Germany) (Burns et al, 1991). The activated BSA solution and the reduced peptide were combined (molar ratio 1:20) and coupling was performed at room temperature for one hour.…”

Section: Production Of Antibodiesmentioning

confidence: 99%

Expression of 11β-hydroxysteroid-dehydrogenase 2 in Sertoli cells of boar testes

Claus

Lacorn

Welter

et al. 2007

Molecular and Cellular Endocrinology

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Abstract11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) activity occurs in boar testes but it is not known which cell types express 11ß-HSD2 mRNA and protein. Therefore, testes samples were taken from mature boars. For immunocytochemistry and Western Blot pig specific antibodies were raised against a 10 amino acid peptide corresponding to amino acids 391-400 of the coding sequence. Quantitative PCR was performed in testis homogenates and additionally RT-PCR in samples collected by UV-single cell microdissection. Data show that in interstitial tissue 11ß-HSD2 is expressed in Leydig cells and additionally in blood capillaries. In tubuli, 11ß-HSD2 primarily is formed in Sertoli cells whereas occurrence in spermatogonia could not be definitely proven. Because glucocorticoid receptors were never found in boar Leydig cells it is concluded that the expression of 11ß-HSD2 in several types of cells forms consecutive lines of defense to protect spermatogonia against glucocorticoidinduced apoptosis.

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“…[15] We envisioned that a photoactivatable trialkylphosphine would be an effective inducer of intracellular reductive stress. Very recently, the first photoactivatable triarylphosphines were reported in the context of light-activated, Bertozzi-Staundiger ligations.…”

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confidence: 99%

Development of a Photoactivatable Phosphine Probe for Induction of Intracellular Reductive Stress with Single‐Cell Precision

Tirla

Rivera‐Fuentes

2016

Angew Chem Int Ed

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Photoactivatable phosphines that induce intracellular reductive stress are reported. The design of these probes takes advantage of the conjugate addition of trialkylphosphines to carbocyanine dyes, which can be reverted photochemically to produce the trialkylphosphine and a fluorescent reporter. The photochemical release depends on the efficiency of photoinduced electron transfer from the indolenine arm of the probe to the coumarin acceptor. These probes readily permeate the mammalian plasma membrane and can be photoactivated in live cells. Upon irradiation of the probe, the released trialkylphosphine induces intracellular reductive stress, which ultimately leads to formation of thioflavin‐positive intracellular protein aggregates. These effects could be induced in individual cells within a monolayer, with minimal disturbance of neighboring cells.

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Selective reduction of disulfides by tris(2-carboxyethyl)phosphine

Cited by 650 publications

References 2 publications

Disulfide structures of highly bridged peptides: A new strategy for analysis

Disulfide structures of highly bridged peptides: A new strategy for analysis

Expression of 11β-hydroxysteroid-dehydrogenase 2 in Sertoli cells of boar testes

Development of a Photoactivatable Phosphine Probe for Induction of Intracellular Reductive Stress with Single‐Cell Precision

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