Practical Protein Chemistry - A. Darbre 1989

Chemical fragmentation of polypeptides
Cleavage of disulfide bonds
Reduction and S-carboxymethylation

Cystine residues can form both intra- and interchain bonds. Therefore, an essential step in sequencing is the Cleavage of disulfide Bonds, primarily to separate The polypeptide chains. At the same time, Disulfide Bonds are highly reactive, which can complicate structural analysis. Consequently, upon cleaving S—S groups, Cysteine residues must be converted into stable derivatives. Finally, following the reduction of cysteine residues, Proteins become more accessible to Proteolytic Enzymes. Methods for cleaving disulfide bonds are discussed in Ch. 4, and A number of reviews on this topic are also available [26, 59, 98, 116]. This chapter includes only those techniques that have found Structure/182.html">Practical Application.

Reduction is among the most selective and practical methods for cleaving S—S bonds. A typical Procedure involves treating the protein with an excess of a low-molecular-weight thiol [equation (2.1)]. Commonly used thiols include cysteine, reduced Glutathione, 2-mercaptoethylamine, thioglycolic acid, 2-mercaptoethanol [78–80, 203], and dithiothreitol (DTT) [29]. The reduction is carried out under an inert gas atmosphere in a slightly alkaline medium in the presence of 8 M urea or 6 M guanidine-HCl. It is worth noting that prolonged storage of urea solutions leads to The formation of cyanate ions, which cause protein modification [182]. It is recommended to use pre-recrystallized urea preparations, and freshly prepared solutions should be deionized using a mixed-bed ion exchanger.

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After removing the reducing agent, thiol groups are readily oxidized by atmospheric oxygen, leading to the reformation of S—S bonds. Therefore, various blocking groups are used to protect SH-groups, yielding stable cysteine derivatives directly in the reaction mixture where the reduction was performed. Iodoacetic acid is most frequently employed as the SH-reagent. This converts cysteine residues into S-carboxymethylcysteine, which remains quite stable during subsequent operations, including sequencing. The degree of modification is determined by Amino acid analysis, taking advantage of the stability of S-carboxymethylcysteine under conditions of acid Hydrolysis. Simultaneously, this allows for the determination of potential losses of Other Amino Acids (Methionine, Tyrosine, Histidine) resulting from Side Reactions [71].

It has been shown that in an acidic medium, S-carboxymethylcysteine (1) can undergo intramolecular cyclization to form a substituted thiazine (2), which cannot be detected by amino acid analysis due to the loss of the amino group [15]. The rate of this reaction depends on Temperature and pH. The optimal conditions for cyclization correspond to pH 3.0 (citrate buffer) and a temperature of 110 °C, whereas under standard acid hydrolysis conditions (6 M HCl, 110 °C, 22 h) S-carboxymethylcysteine is entirely stable. To prevent the loss of S-carboxymethylcysteine through cyclization, it is recommended to alkylate the SH-groups with 3-bromopropionic acid [15].

A typical procedure for reducing the disulfide bonds of Ribonuclease with 2-mercaptoethanol followed by carboxymethylation is described in [3, 35, 102]. Currently, dithiothreitol (3) is the most widely used reducing agent [106]. (This reagent—Cleland's reagent—was first applied for disulfide reduction by Cleland [29].)

The reaction proceeds almost quantitatively, in accordance with equations (2.2) and (2.3), owing to the formation of a six-membered ring (5) that is energetically more favorable than mixed disulfides. At pH 7 and 25 °C, the Redox Potential of dithiothreitol is —0.33 V (compared to —0.22 V for cysteine); hence, the Equilibrium Constant for the overall reduction of cysteine by dithiothreitol is 10⁴. Due to its lower redox potential and resistance to atmospheric oxygen, dithiothreitol can be added at much lower concentrations compared to other thiols. An additional advantage of using dithiothreitol is the near-total absence of an unpleasant odor.

2.2.1.1. Reduction and S-carboxymethylation. Procedure [200]. A protein solution (10–20 mg/mL) in 0.2–0.5 M Tris-HCl buffer (pH 8.0) containing 6 M guanidine-HCl and 2 mM EDTA is sparged with nitrogen in a polypropylene tube, tightly capped, and incubated at 37–50 °C for 30 min. Then, a 50-fold molar excess (relative to the S—S group) of DTT is added—or 300 mol/mol of protein if the number of S—S bonds is unknown—sparged with nitrogen again, and incubated at 37 °C for 4 h.

The reaction mixture is cooled to 0 °C, a slight excess (relative to the SH-group) of recrystallized iodoacetic acid or iodoacetamide is added, and the mixture is incubated in the dark. The pH of the medium is maintained at 8.0 throughout the reaction. After 1 h, the excess reagent is neutralized with 2-mercaptoethanol, and the solution is dialyzed at an alkaline pH and low temperature. These dialysis conditions prevent methionine modification, which can readily occur if dialysis is performed against an acidic buffer. For Desalting, Gel filtration can be performed in a suitable buffer (on Sephadex G-10 or Bio-Gel P-10 in 0.01 M NH₄HCO₃).

2.2.1.2. Reduction with tributylphosphine. Tributylphosphine is an efficient and selective reducing agent for disulfide bonds in keratin (wool) [119]. Studies on the reduction of proteins with known structures demonstrated [152] that the reaction proceeds stoichiometrically According to the equation:

R₁SSR₂ + Bu₃P + H₂O → R₁SH +Bu₃PO      (2.4)

Here, the excess of the reducing agent relative to the S—S groups was 5–20%. In an alkaline medium, the reaction proceeds rapidly, selectively, and almost quantitatively. Alkylation of the resulting SH-groups can be carried out in the presence of the reducing agent. In an acidic medium, the reaction is slow (reaction time 24–48 h). A homogeneous medium is achieved by adding 1-propanol (up to a 1:1 ratio), which further facilitates protein solubilization. Tributylphosphine is toxic and has an unpleasant odor; work with it must be carried out in a fume hood. It is recommended to store the reagent and prepared solutions under nitrogen; a 2% (v/v) solution in propanol has a reducing equivalent of 55–70 µmol/mL.

The procedure proposed in [152]. Bovine pancreatic ribonuclease (64 mg, 5 µmol) is dissolved in 7.5 mL of 7 M urea in 0.1 M Tris-HCl (pH 8.2) containing 1-propanol (2:1). While stirring under a nitrogen atmosphere, 50 µL (130–180 µmol, a 6–9-fold excess) of tributylphosphine is added. After 2 h, 140 µL of 1 M iodoacetate (1 M iodoacetic acid in 1 M NaOH, a 3.5-fold excess relative to SH groups) is added, and the mixture is incubated in the dark. After 5 min, the pH is adjusted to 8.2 with 1 M Na₂CO₃; after 30 min, 0.5 mL of glacial acetic acid is added, and the mixture is lyophilized.

Disulfide bonds can be reduced using Other Reagents; however, these methods find no application in analytical Protein Chemistry. For example, reduction with sodium borohydride (at pH 7–10) is accompanied by peptide bond cleavage [35]; reaction with diborane leads to the reduction of carboxyl groups (in the form of carboxylate ions) to hydroxyl groups [5, 6]. In principle, proteins with unprotonated carboxyl groups can be reduced using diboranes. Electrolytic reduction at a dropping mercury electrode is rarely used in protein chemistry [27, 111].