Assay of leucine aminopeptidase activity in vitro using large-pore reversed-phase chromatography with fluorescence detection.
A chromatographic method for determination of leucine aminopeptidase (LAP) activity in complex matrices is described. L-Leucine-beta-naphthylamide was used as the substrate and its hydrolytic product, beta-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 angstroms) reversed-phase C4 column (RPC4) 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-noise (S/N) ratio with 5 microl sample injection. The method showed a wide dynamic range for quantitation of both the hydrolytic product (10 ng/ml to 80 microg/ml) and LAP (0.1-46.0 microg/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 microg/ml), and 1.6 pmol/(microl min) (17.5 microg/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.
Xiong
Xiang
X
Singapore Eye Research Institute, 11 Third Hospital Avenue, Singapore 168751, Singapore. seri_xx@hotmail.com
Barathi
Amutha
A
Beuerman
Roger W
RW
Tan
Donald T H
DT
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D005260:Female; D007931:Leucyl Aminopeptidase / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008807:Mice, Inbred BALB C; D011817:Rabbits; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
D007931:Leucyl Aminopeptidase