Assay of leucine aminopeptidase activity in vitro using large-pore reversed-phase chromatography with fluorescence detection

摘要

A chromatographic method for determination of leucine aminopeptidase (LAP) activity in complex matrices is described. L-Leucine-β-naphthylamide was used as the substrate and its hydrolytic product, β-naphthylamine, was monitored by fluorescence at 280 nm excitation and 400 nm emission wavelengths. Under optimized conditions, the components in the incubation mixture were baseline separated and eluted out of a large-pore (300 A) reversed-phase C_4 column (RPC_4) within 15 min with a non-linear gradient elution of methanol (0.05% (v/v) trifluoroacetic acid additive). The detection limit of the hydrolytic product reached 0.35 pmol at three time signal-to-nose (S/N) ratio with 5 μl sample injection. The method showed a wide dynamic range for quantitation of both the hydrolytic product (10 ng/ml to 80 μg/ml) and LAP (0.1-46.0 μg/ml) with correlation coefficient larger than 0.998 and reproducibility < 3 and 10% R.S.D. (n = 3), respectively. A fairly broad range of incubation time could be selected within 1 h. The LAP activities and concentrations in rabbit serum, tears, and mouse lens homogenates were determined to be 41.8 (0.3 mg/ml), 2.8 (40.0 μg/ml), and 1.6 pmol/(μl min) (17.5 μg/ml), respectively, with reproducibility of 2-9% R.S.D. (n = 3) and intra-and inter-day variation for the retention time of the hydrolytic product being < 1% R.S.D. (n = 3). The results indicate that the present method is rapid and sensitive as compared to the conventional one.