Practical Protein Chemistry - A. Darbre 1989

Determination of Amino Acid Sequence Using 4-Dimethylaminoazobenzene-4'-isothiocyanate (Manual Procedure)
Reagents and Solvents

DABITC was synthesized as described previously [8, 9] or obtained commercially from Fluka and Pierce. FITC (Merck) was distilled under nitrogen at reduced pressure (b.p. ≈ 92 °C / 12 mm Hg). Pyridine (Merck) was distilled three times: 1) over KOH (10 g/L), 2) with ninhydrin (1 g/L), and 3) over KOH (10 g/L). Trifluoroacetic acid (Fluka) was distilled over CaSO₄∙0,5H₂O (10 g/L). Alternatively, sequencing-grade pyridine and TFA from Pierce can be used.

Polypeptide: 1–10 nmol. Add 80 µL of 50% aqueous pyridine and 40 µL of DABITC in pyridine (10 nmol/µL). Incubate under nitrogen at 52 °C for 50 min. Add 10 µL of FITC and incubate for an additional 30 min.

Extract four times with 500 µL of a heptane – ethyl acetate (2:1) mixture. Evaporate to dryness in a vacuum or under a stream of nitrogen.

Add 50 µL of anhydrous TFA. Incubate (under nitrogen) for 15 min at 52 °C. Remove the solvent by evaporation in a vacuum or under a stream of nitrogen.

Add 50 µL of Water. Extract the thiazolinones with 200 µL of butyl acetate and evaporate the extract in a vacuum to convert them into DABTH-Amino Acid Derivatives.

Subject the residue to the next degradation cycle.

FIG. 14.1. Scheme 1. Reaction sequence in a single cycle of liquid-phase degradation using the DABITC – FITC reagent.

Other analytical-grade Solvents were used without further purification.

Polyamide sheets from Schleicher and Schüll or Cheng-Chin Co. were used for identification. Amination of Glass beads (CPG-75, Serva) and their activation with *p*-phenylene diisothiocyanate (DITC) were performed as described in Chapter 12 [1].