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NDM-1 activity assay

HS Hongzhe Sun
QZ Qi Zhang
RW Runming Wang
HW Haibo Wang
YW Yuen-Ting Wong
MW Minji Wang
QH Quan Hao
AY Aixin Yan
RK Richard Yi-Tsun Kao
PH Pak-Leung Ho
HL Hongyan Li
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The NDM-1 activity assay was performed based on a previous reported method34. Briefly, NDM-1 or NDM-1-C208A (50 nM) were incubated with Au(PEt3)Cl in enzyme assay buffer [50 mM HEPES buffer, 100 mM NaCl at pH 7.4] for 1 h at 25 °C, and then mixed with equal volume of 200 μM nitrocefin. The assay was performed in 96-well plate using the kinetic mode on a Varian Cary50 UV-visible spectrophotometer at 25 °C. The increase in absorbance at 490 nm was monitored every minute for a duration of 30 min until the reaction was completed. Linear portions of curves were used for data analysis. For the time-dependent incubation assay, NDM-1 (10 nM) were preincubated with Au(PEt3)Cl (5 µM) for different time from 0 to 90 min, followed by the enzyme activity measurement using the method mentioned above.

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