Practical Protein Chemistry - A. Darbre 1989

Methods of solid-phase amino acid sequence analysis
Attachment methods
DITC method using isothiocyanate glass

12.4.2.1. Advantages, limitations, and coupling Procedure

Advantages.

1. Coupling of Proteins AND Peptides to Glass-Based Supports is characterized by a higher yield than when using Polystyrene-Based Resins.

2. The common problem of peptide solubility in the coupling buffer is more easily resolved. If the peptide dissolves poorly in the standard buffer, pyridine and/or DMF can be added before introducing the support. During the coupling of a DITC-activated peptide to an amino-containing support, the reagent shows varying tendency to precipitate as a urea derivative, depending on the Water content required to dissolve the peptide.

3. The method is rapid to perform.

Limitations. The method is applicable only to peptides containing Lysine or aminoethylcysteine residues. Peptides containing these Amino Acids within the chain can only be analyzed up to the specified residues, which serve as attachment points. The method cannot be recommended for arginyl-containing peptides.

Coupling procedure [40, 41, 42, 62, 72]. Peptide-to-support ratio: 1 nmol per mg of glass. Dissolve 10–200 nmol of peptide or protein in 30–100 µl of buffer No. 1 under nitrogen. Check pH and solubility (Section 12.4.5). Add 10–100 mg of DITC-APS (dry) and stir under nitrogen for 60 min at 30 °C. Add 50–100 µl of ethanolamine and stir under the same conditions. Wash the reaction mixture thoroughly with DMF, methanol, and ether (twice with 2 ml of each solvent). The supernatant is monitored for peptide content; the support is dried in vacuo.

Modified procedure. If the peptide is insoluble in the N-methylmorpholine-based buffer, coupling can be performed in one of the following systems: 50% aqueous pyridine; DMF added after The addition of triethylamine (buffer No. 3) or N-methylmorpholine; sodium bicarbonate (pH 9.0; buffer No. 4) with the addition of 1% SDS (if necessary). It was found that during the coupling of the Insulin B-chain in N-methylmorpholine-TFA buffer, pH 9.0, the yield is 45–50%, whereas in sodium bicarbonate buffer (pH 9.0) it is 66–70% [11].