DOUBLE ISOTOPE ASSAY OF NANOGRAM QUANTITIES OF PEPTIDES AS THEIR DINITROPHENYL DERIVATIVES - ANGIOTENSIN AND RELATED COMPOUNDS

A double-isotope derivative assay has been developed which allows quantitation of angiotensin II on a nanogram scale. The peptide is reacted with 3H-labeled 1-fluoro-2,4 dinitrobenzene (3H-FDNB) to yield a trisubstituted dinitrophenyl (DNP) derivative under conditions in which it has been shown that reaction of the peptide with FDNB is quantitative. Separately prepared 14C-DNP-angiotensin is added as indicator and nonlabeled DNP peptide as carrier. Excess reagent is removed by extraction with toluene, and the peptide is extracted into ethyl acetate and subjected to TLC on silica gel. The yellow DNP derivative is eluted, converted to its methyl ester with methanol-HCl, rechromatographed, eluted, and its 3H and 14C radioactivity determined in a liquid scintillation spectrometer. The reagent blank is zero. Yields of derivative are quantitative. Recoveries are variable (20-40%) but accurate correction is provided by use of the 14C indicator. With 3H-FDNB of specific activity of 200 μC/μm two or more nanograms can be accurately determined. Angiotensin II has also been assayed on a nanogram scale after carboxypeptidase degradation to its des-phenylalanine heptapeptide, using appropriate heptapeptide 14C indicator and carrier. The DNP derivatives of several peptides related to angiotensin I and II have been prepared and shown to be separable by TLC. The 3H-DNP derivative method should be readily adaptable to the measurement of a variety of other peptides, including angiotensin I, and may provide the basis for an assay of renin in plasma. © 1969.