Protein Protocols Protein Protocols

Table 1
Summary of Methods for Cleavage of Trp-X Bonds in Polypeptides
Brief method detailsa Comments Example ref.
1. Incubation in molar excess of cyanogen bromide: See text for further details. 4
a. Incubation in glacial acetic acid: 9 M HCl (2/1 [v/v] Yields and specificity excellent; Met oxidized to sulfoxide by step a.
with DMSO, room temperature, 30 min.
b. Neutralization
c. Incubation with CNBr in acid (e.g., 60% formic acid)
4°C, 30 h in the dark.
2. Incubation in very large (up to 10,000-fold) molar To inhibit Met-X cleavage, Met is photooxidized irreversibly to 8
excess of CNBr over Met, in heptafluorobutyric Met sulfone; yield poor.
acid–88%–HCOOH (1/1 [v/v]), room temperature,
24 h in the dark.
3. a. Incubate protein for 30 min, room temperature, in: Cys and Met oxidized; some deamidation and cleavage around 9
21.6 mg/mL of phenol in glacial acetic acid 12 M Asp may occur; fresh, colorless HBr required.
HCl–DMSO:24:12:1,
b. Add 0.1 vol of 48% HBr, 0.03 by volume of Trp converted to dioxindolylalanyl lactone. Protein containing
DMSO, and incubate for 30 min, room temperature. dioxindolylalanine derivatives(s) but remaining uncleaved
may be cleaved to improve yield to approx 80% as follows: Incubate
in 10% acetic acid, 60°C 10–15 h.
4. Incubate in BNPS-skatole b 100-fold molar excess Reagent is unstable—fresh reagent required to minimize side 10
over Trp) in 50% acetic acid (v/v) room temperature, reactions; some reactions with Tyr may occur, but addition of
48 h, in the dark. free Tyr to reaction mixture minimizes this; Met and Xys
may be oxidized, yields up to 60%.
494 Smith