Application of a coupled reducing system to Edman sequencing.

Addition of low levels of solid tris-(2-carboxyethyl)-phosphine hydrochloride to Applied Biosystems' R4A (2 mM) and R5 (0.17 mM) resulted in a coupled, phosphinedithiothreitol reducing system. Coupling the two reductants eliminated or greatly decreased interference from the oxidation product of dithiothreitol and allowed quantitation of phenylthiohydantoin (PTH)-Asp below the 400-fmol level. Use of the system resulted in a measurable increase in repetitive yield for beta-lactoglobulin, and instrument PTH standards were stable for more than 3 months.