Please use this identifier to cite or link to this item:
|Title:||Assay of leucine aminopeptidase activity in vitro using large-pore reversed-phase chromatography with fluorescence detection|
Reversed-phase C4 chromatography
|Citation:||Xiong, X., Barathi, A., Beuerman, R.W., Tan, D.T.H. (2003-10-25). Assay of leucine aminopeptidase activity in vitro using large-pore reversed-phase chromatography with fluorescence detection. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences 796 (1) : 63-70. ScholarBank@NUS Repository. https://doi.org/10.1016/j.jchromb.2003.08.005|
|Abstract:||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 280nm excitation and 400nm emission wavelengths. Under optimized conditions, the components in the incubation mixture were baseline separated and eluted out of a large-pore (300Å) reversed-phase C4 column (RPC4) within 15min with a non-linear gradient elution of methanol (0.05% (v/v) trifluoroacetic acid additive). The detection limit of the hydrolytic product reached 0.35pmol at three time signal-to-noise (S/N) ratio with 5μl sample injection. The method showed a wide dynamic range for quantitation of both the hydrolytic product (10ng/ml to 80μg/ml) and LAP (0.1-46.0μg/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 1h. The LAP activities and concentrations in rabbit serum, tears, and mouse lens homogenates were determined to be 41.8 (0.3mg/ml), 2.8 (40.0μg/ml), and 1.6pmol/(μlmin) (17.5μg/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. © 2003 Elsevier B.V. All rights reserved.|
|Source Title:||Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences|
|Appears in Collections:||Staff Publications|
Show full item record
Files in This Item:
There are no files associated with this item.
checked on May 17, 2018
WEB OF SCIENCETM
checked on May 9, 2018
checked on May 18, 2018
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.