Determination of Inhibitory Effects of Inhibitors on the Tumor Necrosis Factor-αConverting Enzyme Using High-Performance Liquid Chromatography / 华中科技大学学报(医学版)

ABSTRACT

Objective A high-performance liquid chromatography(HPLC)method was modified and used to determine the inhibitory effects of GM6001 and inhibitor A on tumor necrosis factor-α converting enzyme(TACE).Methods TACE and polypeptides substrate were incubated for 15 min at 37°C.Ala-Dpa was added as internal standard of quantitative analysis.Then the solution was analyzed by HPLC.The 55% methanol aqueous solution was used as the mobile phase.The wavelength of detector was 353 nm.The ratio of the peak area of remaining substrate to that of internal standard was determined.And the amounts of inverted substrate could be obtained from calibration curve.The TACE activity could be calculated.Results The relative peak areas of substrate were linearly increased depending on the growth of substrate concentration.The correlation coefficient was 0.996 8 and linear range was from 10 to 400 μmol/L.Precision experiments indicated that the precision was improved obviously by using internal standard method in the determination of TACE activity by HPLC.The values of 50% inhibitory concentration IC50 of GM6001 and inhibitor A determined by the newly proposed method were 317.5 and 175.8 nmol/L,respectively.Conclusion The HPLC method assaying TACE activity with Ala-Dpa as internal standard is more accurate,and more practical for screening of TACE inhibitors.