Practical Protein Chemistry - A. Darbre 1989

Polypeptide Fragmentation by Chemical Methods
Cleavage at Tyrosine Residues
Cyanylation with 2-Nitro-5-thiocyanobenzoic Acid

The protein sample is dissolved in 0.2 M Tris-acetate buffer (pH 8) containing 6 M guanidine-HCl. If the protein contains no S—S groups, a slight excess of dithiothreitol is added, and the mixture is incubated at 20 °С for 30 min. To reduce S—S groups, a 10 mM concentration of dithiothreitol is established, followed by incubation at 37 °С for 1—2 h. Next, 2-nitro-5-thiocyanobenzoic acid is added (a 5-fold molar excess relative to the SH group); if necessary, the pH is maintained using NaOH, and the mixture is incubated at 37 °С for 15 min. The reaction mixture is acidified to pH≤4, cooled to 4 °С, and low-molecular-weight components are removed by dialysis or Gel filtration in 50% acetic acid or another volatile low-pH buffer. The sample can be stored in 50% acetic acid at —20 °С. To carry out the Cleavage reaction, the sample is thoroughly dried, redissolved in 0.1 M Na-borate buffer (pH 9.0) containing 6 M guanidine-HCl, and incubated at 37 °С for 12—16 h.

The widespread application of the Cysteine-specific cleavage method for generating large fragments is hindered by the fact that the C-terminal fragment is blocked at the amino group by an iminothiazolidine residue. Attempts at deblocking using classical Edman Degradation yielded negative results [96]. Consequently, the resulting Peptides, with the exception of the N-terminal one, cannot be used for direct sequencing. A method for modifying these peptides using Raney nickel was recently proposed [133]. Upon treating the protected peptides with active (neutral) W-6 catalyst in a neutral medium at 50 °С for 6—10 h, mild desulfurization occurs, yielding N-alanyl Peptides with a 90% yield [9]. During the reaction, cysteine and Methionine are rapidly converted into Alanine and a-aminobutyric acid, respectively.

2.7.1.1. Procedure [133]. Desulfurization is carried out in 0.05 M Tris-HCl buffer (pH 7) at 50 °С. Raney nickel W-6 is prepared [9]. A mixture of 20 mg of peptide and 200 mg of catalyst in 50 mL of buffer solution is heated to boiling for 7 h under a nitrogen atmosphere. The supernatant liquid is filtered through a Column (0.6x5 cm) packed with Chelex-100 chelating resin. The peptides are eluted with 1 M ammonia, and the eluate is lyophilized. Owing to the high yield in both the cleavage and subsequent desulfurization of iminothiazolidine peptides, this procedure is well-suited for Protein Chemistry to generate large fragments.