SHORT COMMUNICATIONS A Simplified Method for Chromatography of Dnp-Leucine in the Determination of Intracellular Specific Activity of [ 3H] Leucine
Regier and Kafatos (1) have recently described a sensitive and convenient microtechnique for determination of the intracellular specific activity of [ 3H ]Leu in small quantities, using ["Cl fluorodinitrobenzene and two-dimensional thin layer chromatographic separation of the Dnp-(amino acids). We found this procedure attractive, but we required simultaneous analyses of a rather large number of samples (which is inconvenient on two-dimensional chromatography), so we tried a series of alternative separation procedures. We were delighted to find that benzene:pyridine:glacial acetic acid (80:20: 1) [the "benzene" system of Brenner et al. (2) used in the second dimension by Regier and Kafatos] gave useful separation of Dnp-Leu from other Dnp-(amino acid)s in the mixture without any prior chromatography.
We prepared ['"Cl Dnp-(amino acid) s from the acid-soluble fraction of mouse hearts as described by Regier and Kafatos (1). Routinely, 50 ~1 of the neutralized trichloroacetic acid supernate was treated with 10 ~1 ['"Cl fluorodinitrobenzene (Amersham-Searle, 0.4 #Zi/pmole, dissolved in ethanol at 3.2 mg/ml). To facilitate monitoring the chromatographic separation, 1.0 pg each of unlabeled Dnp-Leu, Dnp-Ile, and Dnp-Val (Sigma) were added to the final [14C]Dnp-(amino acid) mixture. The ether solutions of Dnp-(amino acid) s were spotted at the origins of silica gel thin-layer chromatography plates (20 X 20 cm, 0.1 mm thick, Eastman No. 6060), and the chromatograms were developed by continuous horizontal chromatography for 5 hr at room temperature under windless conditions. All solvents were purified as described by Stahl (3). The chromatography apparatus was prepared from ordinary double-strength window glass essentially as described by Brenner and Niederwieser (4) ; 266