[10] Reductive cleavage of cystine disulfides with tributylphosphine

Rüegg, Urs Th.; Rudinger, J.

doi:10.1016/0076-6879(77)47012-5

Cited by 245 publications

(126 citation statements)

References 13 publications

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“…In contrast to exchange-reduction by thiols, trialkylphosphines reduce disulfides irreversibly and stoichiometrically (Ruegg & Rudinger, 1977):…”

Section: Reduction Of Disulfides By Trialkylphosphinesmentioning

confidence: 99%

“…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.…”

Section: Reduction Of Disulfides By Trialkylphosphinesmentioning

confidence: 99%

“…Reduction by agents such as mercaptoethanol or dithiothreitol actually proceeds via the exchange reaction and thus will not occur at low pH. 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.…”

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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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“…In contrast to exchange-reduction by thiols, trialkylphosphines reduce disulfides irreversibly and stoichiometrically (Ruegg & Rudinger, 1977):…”

Section: Reduction Of Disulfides By Trialkylphosphinesmentioning

confidence: 99%

Section: Reduction Of Disulfides By Trialkylphosphinesmentioning

confidence: 99%

mentioning

confidence: 99%

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

1993

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“…As an alternative, we have tested the ability of Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to reverse the effect of Cu (II) Phe. The mechanism of action of TCEP is different from that of DTT, and TCEP, unlike DTT, is highly specific for reducing disulfide bridges (27). Fig.…”

Section: Disulfide Bridges Are Formed Between Cysteines At Position 3mentioning

confidence: 99%

Depolarization Induces Intersubunit Cross-linking in a S4 Cysteine Mutant of the Shaker Potassium Channel

Aziz¹,

Partridge²,

Munsey

et al. 2002

Journal of Biological Chemistry

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Voltage-gated potassium (K v ) channels are integral membrane proteins, composed of four subunits, each comprising six (S1-S6) transmembrane segments. S1-S4 comprise the voltage-sensing domain, and S5-S6 with the linker P-loop forms the ion conducting pore domain. During activation, S4 undergoes structural rearrangements that lead to the opening of the channel pore and ion conduction. To obtain details of these structural changes we have used the engineered disulfide bridge approach. For this we have introduced the L361C mutation at the extracellular end of S4 of the Shaker K channel and expressed the mutant channel in Xenopus oocytes. When exposed to mild oxidizing conditions (ambient oxygen or copper phenanthroline), Cys-361 formed an intersubunit disulfide bridge as revealed by the appearance of a dimeric band on Western blotting. As a consequence, the mutant channel suffered a significant loss in conductance (measured by two-electrode voltage clamp). Removal of native cysteines failed to prevent the disulfide formation, indicating that Cys-361 forms a disulfide with its counterpart in the neighboring subunit. The effect was voltage-dependent and occurred during channel activation after Cys-361 has been exposed to the extracellular phase. Although the disulfide bridge reduced the maximal conductance, it caused a hyperpolarizing shift in the conductance-voltage relationship and reduced the deactivation kinetics of the channel. The latter two effects suggest stabilization of the open state of the channel. In conclusion, we report that during activation the intersubunit distance between the N-terminal ends of the S4 segments of the L361C mutant Shaker K channel is reduced.

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“…In the case of insulin, the disulphide bonds were first reduced with tributylphosphine as previously described (Ruegg & Rudinger, 1974a, 1977a. co-Toluenesultone was found to react specifically and in high yield with the thiol groups so generated.…”

Section: Resultsmentioning

confidence: 99%

2-Sulphobenzyl, a new solubilizing and reversible protecting group for cysteine in proteins. Its scope and limitations

Rüegg¹,

Jarvis²,

Rudinger³

1979

Biochemical Journal

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S-2-Sulphobenzylcysteine and S-2-(sulphomethyl)benzylcysteine are prepared by alkylation of cysteine with omega-toluenesultone and 2,3-benzo-1,4-butanesultone respectively. Owing to the presence of the sulphonic acid group, these protected cysteine derivatives are extremely water-soluble and are stable to acid hydrolysis. The groups can be removed by treatment with sodium in liquid NH3. Reduction with tributylphosphine and simultaneous alkylation of insulin with toluenesultone under mild conditions (pH 8.3, aq. 50% propanol) gives the fully S-substituted derivatives in excellent yield; they can be separated by isoelectric precipitation of the S-sulphobenzylated B-chain. Treatment of the latter with sodium in liquid NH3 led simultaneously to the removal of the protecting groups and to the well-documented cleavage at the threonine-proline bond which can be prevented by addition of sodium amide. When deprotected A-chain was recombined with B-chain, insulin was isolated in the same yield and with the same degree of biological activity as that in the control experiment.

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[10] Reductive cleavage of cystine disulfides with tributylphosphine

Cited by 245 publications

References 13 publications

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

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

Depolarization Induces Intersubunit Cross-linking in a S4 Cysteine Mutant of the Shaker Potassium Channel

2-Sulphobenzyl, a new solubilizing and reversible protecting group for cysteine in proteins. Its scope and limitations

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