Practical Protein Chemistry - A. Darbre 1989

Polypeptide Fragmentation by Chemical Methods
Cleavage at Tryptophan Residues
Other Reagents

Tryptophan Cleavage can be carried out via Oxidative Halogenation using "active iodine" generated by the hydrogen peroxide – iodide salt – lactoperoxidase (or horseradish peroxidase) system. Simple Peptides containing a tryptophan residue are cleaved at pH 5 within 10 min with a 30–40% yield. The presence of all components of this system is required for the reaction to proceed. Tryptophan cleavage proceeds with a similar yield using other iodinating agents, such as І₂, І₃^-, ІСl, and chloramine-T/KI [1]. The feasibility of using peroxidase-assisted halogenation for Protein Cleavage remains an open question, yet the method warrants attention.

2.5.6.1. Tryptophan cleavage by N-chlorosuccinimide. Peptide bonds at the carboxyl terminal of tryptophan are selectively cleaved in an acidic environment by N-chlorosuccinimide, while other peptide bonds remain unaffected. Model compound experiments have shown that both the yield and the reaction rate are somewhat lower here compared to N-Bromosuccinimide. However, the reaction proceeds with high selectivity, and no signs of cleavage at Tyrosine or Histidine residues are observed. Tryptophan peptide bonds in $\alpha$-lactalbumin, the Trypsin inhibitor (Kunitz inhibitor), Myoglobin, and Glucagon are cleaved by N-chlorosuccinimide in aqueous acetic acid with a 19–58% yield [172]. Some studies [115] recommend adding urea to the reaction mixture (this specific protocol is detailed below).

Procedure. The protein concentration in the reaction mixture is 0.286 mmol/L, and the protein-to-N-chlorosuccinimide molar ratio varies depending on the subject of study. The working buffer contains 27.5% acetic acid and 4.68 M urea. The reaction is carried out at 20 °C for 30 min and quenched by The addition of N-acetyl-L-Methionine [172]. According to the standard procedure, the protein sample is dissolved in distilled Water (1 nmol/mL) and mixed (in a 4:10 volume ratio) with the N-chlorosuccinimide buffer solution (1 mL of glacial acetic acid + 1 g of urea + 1 mL of water).

In cytochrome c samples from various sources, tryptophan cleavage proceeds with a yield of ~50%.

2.5.6.2. Cleavage using tribromocresol [137]. Selective Cleavage of Proteins AND PEPTIDES at tryptophan peptide bonds is carried out using 2,4,6-tribromo-4-methylcyclohexadienone (tribromocresol)

(59). Peptide bonds of tyrosine and histidine, which are typically cleaved by brominating Reagents such as N-bromosuccinimide, remain unaffected under these conditions. However, tyrosine is converted to its 3,5-dibromo derivative, Cysteine is oxidized to cysteic acid, methionine to methionine-S-oxide, and histidine residues undergo modification. The optimal conditions for tryptophan cleavage are as follows: 3 eq. of tribromocresol, pH 3, 20 °C, 5–15 min. In the case of Lysozyme, selective cleavage proceeds with a 5–60% yield. It is generally accepted that the reaction proceeds via an oxidative halogenation mechanism, similar to that of N-bromosuccinimide.

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Procedure. To a solution of glucagon (0.5 \mu mol) in 2 mL of 70% acetic acid, 25 \mu mol of tribromocresol in 50 \mu L of dioxane is added. The mixture is incubated with stirring for 15 min and then chromatographed in 0.1 M acetic acid on a Sephadex G-25 Column.