Practical Protein Chemistry - A. Darbre 1989

Determination of the amino acid sequence of peptides by the Edman method with identification of dansyl amino acids
Edman condensation and cleavage

For the Edman Degradation reaction (Fig. 10.1) to proceed successfully, the following conditions are required: The Use of pure Reagents, the exclusion of oxygen from the reaction environment, and thorough drying of the Peptides following the Condensation and Cleavage steps. Reagents must be freshly distilled [2], and the condensation reaction mixture saturated with nitrogen. Peptide samples are pre-dried meticulously in vacuum desiccators heated within metal blocks.

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FIG. 11.3. Approximate positions of dansyl amino acid spots (hatched spots) relative to marker spots (dark spots). Hse — DNS-homoserine, Y⁰ — DNS-Tyr, Y_а⁰ — di-DNS-Tyr, Кε — ε-DNS-Lys, Ка — а-DNS-Lys, К_аε — di-DNS-Lys, КаPTC — а-DNS ε-PTC-Lys, СМС — DNS-CMCys, DNS-OH — dansyl acid, DNS-NН₂ — dansyl amide. Designations for other DNS-Amino Acids are given in the single-letter code.

The Procedure described below is certainly inferior to automated Methods both in analysis speed (no more than two cleavage cycles per day for a single peptide) and in step yields, yet it is highly convenient for the simultaneous structural determination of several short peptides. In this case, the time-consuming process of individually saturating each sample with nitrogen prior to the condensation stage can be optimized. For this purpose, the author utilized a simple device: a manifold featuring 20 needle outlets from syringes (protected against corrosion by Teflon tubing). The needles are arranged so that when lowered into the brass heating block, they enter the sample test tubes. Instead of screw caps, the tubes are tightly sealed with adhesive tape, which is applied simultaneously using a Perspex sheet containing 20 wells. A commercially available device that also provides Temperature control for heating samples in the block is available (the N-Evap by Organomation).

The Edman degradation process requires the following reagents (distilled or sequencing-grade) and equipment: phenylisothiocyanate (10% solution in pyridine, stored in the dark at —20°C for no more than one week); aqueous pyridine (1:1, stored in the dark); trifluoroacetic acid; ethanol (95%); n-butyl acetate; nitrogen gas (in a cylinder); Pyrex test tubes pre-ignited at 500—600°C; a temperature-controlled heating block, vacuum desiccators, and a vacuum line connected to an oil pump; desiccators containing phosphorus pentoxide and sodium hydroxide (for drying PTC-peptides after the condensation stage); and an additional desiccator containing sodium hydroxide (intended for drying peptides after the cleavage stage).