I have been trying to measure POX, PPO and PAL enzyme activity in wheat by using the following procedures:
Wheat leaves will be macerated with a mortar and pestle in the presence of liquid nitrogen, and 10 mL of phosphate-potassium buffer (0.1 mol/L; pH 6.1) will be added to 0.2 g of macerated leaves. After resting for one hour at 4°C and periodical agitation, the solution will be centrifuged at 13,000 g for 15 minutes, at 4°C. Supernatants (enzyme extracts) will be then utilized for enzymatic activity determination within 2h, store on ice till the assay is carried out. Peroxidase (POX) and polyphenoloxidase (PPO) activities will be measured by spectrophotometry through the increase in optical density (OD), using OD470/min/g and OD420/min/g, respectively. The substrates utilized by POX will be 0.2 ml guaiacol (0.01 mol/L) and 0.15 ml H2O2 (0.1 mol/L) solutions placed in a cuvette and by PPO will be 1.0 ml catechol (0.001 mol/L) solution. The readings will be performed each second, for 2 minutes and the activities will be expressed as units per gram of fresh weight (u/g). One activity unit will be defined as the increment of 0.1 absorbance unit per minute. To determine the phenylalanine ammonia-lyase (PAL) activity, the enzyme extract will be prepared with 1 g of macerated leaves, 5 mL tris-HCL (0.1 mol/L buffer; pH 8.8), and 0.1 g insoluble polyvinylpyrrolidone. The buffer will be contained 0.75 ml EDTA (0.001 mol/L; pH 8.8) and 0.75 ml β-mercaptoethanol (0.01 mol/L). After resting for one hour at 4°C and periodical agitation, the solution will be centrifuged at 1,500 g for 5 minutes, at 4°C. The supernatants (enzyme extracts) will be utilized for enzymatic activity determination. PAL activity will be measured by spectrophotometry through the increase in OD280/min/g. The vials containing the reaction mixture will be incubated in water bath at 37°C for five periods of time (0, 30, 60, 90, and 120 minutes), with 0.5 mL perchloric acid (2 mol/L) utilized to halt the reaction. Activity will be expressed as activity units per gram of fresh weight (u/g). One unit will be defined as the amount of trans-cinnamic acid formed per minute under the assay conditions.
I am confused about the quantities, pH and molarities of buffers used in this experiment. Can anyone help me about these 3 things?
To make it simple, prepare 10 mg/mL of substrate in 10 mM buffer and incubate with 1-10 mg protein to measure the activity
I am 1) not sure if it is wise to use 50 fold ratio buffer/leaves for POX assay extraction, i use 10 volumes of buffer and why pH 6.1 ? There was a recent review about ROS experiments i would use a standard phosphor buffer pH 7.8 100 mM with 5 % PVPP and 0,5 % Triton X-100 added.. http://doi.wiley.com/10.1111/pce.12726
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