Practical Protein Chemistry - A. Darbre 1989

Chemical Fragmentation of Polypeptides
Cleavage at Tryptophan Residues
Cleavage at Tryptophan Residues in the DMSO – Hydrohalic Acid System

Tryptophan-containing Proteins AND Peptides can be selectively cleaved using a mixture of DMSO and hydrobromic acid in acetic acid. In terms of reaction product yield, this method is comparable to Cleavage with BNPS-skatole, yet it surpasses the latter in reagent availability and ease of Structure/175.html">Implementation [51, 163]. The reaction mechanism involves the Oxidative Halogenation of the tryptophan indole ring. In fact, the interaction between sulfoxide and hydrohalic acid entails several equilibrium reactions [equation (2.15)], yielding a halosulfonium ion R₁R₂SX⁺ as the main intermediate product [14]. Halogenation of the tryptophan indole ring can be effected by all three intermediates: the sulfide dihalide, the halosulfonium halide, and free halogen.

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While the DMSO–HCl mixture strictly converts tryptophan into oxoindolylalanine [162, 164], the DMSO–HBr mixture oxidizes tryptophan to dioxoindolylalanine with the simultaneous formation of a spirolactone [51]. Tryptophan-containing peptides are also oxidized by the DMSO–HBr mixture to dioxoindolylalanine, and it is only under these conditions that oxidation is accompanied by peptide bond cleavage. Furthermore, it has been demonstrated that oxoindolylalanine peptides obtained by Treatment with the DMSO–HCl mixture can be cleaved upon subsequent treatment with the DMSO–HBr mixture. This fact indicates that an oxoindole is formed as a reaction intermediate [162–164]. To achieve optimal product yields, it is recommended to carry out sequential treatment of proteins with DMSO–HCl and then DMSO–HBr mixtures in situ. Direct interaction of tryptophan-containing peptides with DMSO–HBr leads to The formation of 2-dimethylsulfoniotryptophan (58) as an intermediate. This compound presumably forms via electrophilic substitution at position 2 of the tryptophan indole ring by protonated DMSO, analogously to the reaction observed in the series of phenols and their ethers [63].

The cleavage reaction driven by the DMSO–hydrohalic acid mixture proceeds selectively at the tryptophan peptide bond. Meanwhile, Methionine is oxidized to the S-oxide, and Cysteine to cystine. In practice, a small amount of phenol is added to the reaction mixture to protect Tyrosine residues. It is also advisable to add phenol to 48% HBr to scavenge traces of bromine. These measures are fully sufficient to preserve tyrosine entirely [163], although one exception involving its degradation has been reported [57].

2.5.3.1. Cleavage at the tryptophan residue by treatment with DMSO–HCl and DMSO–HBr mixtures [163]. Cytochrome c (17 mg) and 20 mg of phenol are dissolved at 20 °C in a mixture of the following composition: glacial acetic acid (600 µL) + 12 M HCl (300 µL) + DMSO (12 µL). After 30 min, 48% HBr (100 µL) and DMSO (25 µL) are added to the reaction mixture, and the solution is incubated at 20 °C for 30 min. Then, 2 mL of Water is added, and the mixture is extracted several times with ethyl acetate (to remove the heme and its degradation products). The aqueous phase is partially evaporated in vacuo, and the residue is chromatographed on a Sephadex G-50 (superfine) Column (2 × 14 cm) using 10% formic acid as the eluent. The cytochrome c peptide fragments elute as three fractions. The first fraction, preceded by traces of aggregated protein, contains apocytochrome c with modified tryptophan-59 (in the form of dioxoindolylalanine). The second and third fractions contain fragments 1–59 and 60–104, respectively (the yield of reaction products, ~60%, is determined by weighing after lyophilization).

The fragments are identified by the Amino Acid Composition of the hydrolysate following Hydrolysis with 3 M p-toluenesulfonic acid [117]. The amino acid yields from the analysis matched the composition of apocytochrome c and its fragments. Methionine residues at positions 65 and 80 were oxidized to methionine S-oxide. The reagent brought about the complete detachment of the heme, similarly to the action of BNPS-skatole on cytochrome c [50].

2.5.3.2. Cleavage at the tryptophan residue by the action of DMSO–HCl and BrCN [83]. Cystine (cysteine) residues present in the protein are preliminarily reduced and alkylated using standard Classification Reagents. A small volume of the sample solution containing 2–3 nmol of protein is placed in a 1.5 mL Eppendorf centrifuge microtube and lyophilized. To the dry residue, 4.9 µL of freshly prepared reagent (300 µL of glacial acetic acid + 150 µL of 9 M HCl + 40 µL of DMSO) is added, and the mixture is incubated at 20 °C for 30 min or at 4 °C for 2 h. The reaction mixture is then cooled in an ice bath, followed by The addition of 4.4 µL of ice-cold 15 M (commercial) NH₄OH (protein precipitation may occur at this stage) and 40 µL of a Cyanogen bromide solution (0.3 g/mL) in 5 M acetic acid or 60% formic acid. The tube is tightly closed and sealed with Parafilm. The reaction mixture is incubated in the dark at 20 °C for 12–15 h or at 4 °C for 30 h.

Tryptophan cleavage proceeds almost quantitatively, regardless of The sequence of neighboring Amino Acids. The reaction is not accompanied by the cleavage or modification of Other Amino Acids. The presence of small amounts of salts, ionic detergents, or Coomassie Brilliant Blue does not interfere with the reaction.