摘要：We developed a convenient chromogenic assay method for the activity of sphingomyelinase (SMase) from Bacillus cereus . SMase reaction was quenched by Zn2+, and the released phosphocholine was converted into a choline by the action of alkaline phosphatase. After that, the choline was converted into a chromogenic dye by the actions of choline oxidase and peroxidase in the presence of EDTA to trap the added Zn2+ which could interfere with the choline oxidase/peroxidase reactions. Triton X-100 also was added to the reaction mixture, in order to remove turbidity generated from ceramide which had been produced by the SMase reaction. To test a large number of samples in a short period of time, this assay was performed using 96-well microtiter plates. This method proved to be applicable not only to the measurement of the hydrolysis of sphingomyelin but also to those of lysophosphatidylcholine (lysoPC) and lyso platelet-activating factor by B. cereus SMase. Using this method, the kinetic parameters ( K m and k cat) for B. cereus SMase toward various types of substrates were then determined, and the effect of Triton X-100 on the hydrolysis of lysoPC was examined.
关键词：sphingomyelinase;chromogenic assay;Bacillus cereus;zinc ion
