Phosphate Buffer (0.1M, pH 7.0)
It is prepared by dissolving 1.74g of Dipotassium hydrogen phosphate (K2HPO4) in 100mL Deionized water and 1.36g of Potassium phosphate monobasic (KH2PO4) in 100mL deioinized water. Mix 61.5mL of K2HPO4 and 38.5 mL of KH2PO4 and raise the volume 1 Litre to get 0.1M Phosphate buffer (pH 7.0).
Phosphate Buffer (150mM, pH 7.1)
It is prepared by dissolving 2.61g of K2HPO4 in 100mL Deionized water and 2.04g of KH2PO4 in 100mL deioinized water. Mix 61.5mL of K2HPO4 and 38.5 mL of KH2PO4 and raise 1 Litre to get 150mM Phosphate buffer (pH 7.1)
Preparation of Standard Solution
Ornithine-HCl: Dissolve 1.682mg of L-Ornithine HCl in 10mL of phosphate buffer (pH 7.0). Dilute the stock solution to obtain working solution in the range of 0.2-100μM.
Putrescine Dihydrochloride: Dissolve 1.6107mg of Putrescine Dihydrochloride in 10mL of phosphate buffer (pH 7.0). Dilute the stock solution to obtain working solution in the range of 0.2-100μM.
Reaction Mixture: Dissolve 17.57μL of β-mercaptoethanol (2.5 mM), 55.84mg of Disodium EDTA (1.5mM), 2μL of stock solution of Pyridoxal phosphate prepared in 150mM Phosphate buffer (75nM) and 50.6mg of L-Ornithine HCl (3mM) in 150mM Phosphate buffer (pH 7.1).
Picryl Sulfonic acid (TNBS): 25.7μL of TNBS dissolved in 10mL of 1-Pentanol, freshly prepared and kept at 4ºC
Caution! β-mercaptoethanol is a toxic by inhalation, cause damage to skin and eyes by prolonged exposure, so this chemical bottle should be opened inside fume hood and for very short time. Wear masks and gloves while using it.
0.1M Sodium Borate: Dissolve 3.8137g in 100mL Deionized water, adjust pH (8.0) by 1M either NaOH and/or 1M HCl.
4N sodium Hydroxide: Dissolve 16.0g in 100mL of Deionized water.
1M Perchloric acid: Commercial solution available is calculated as 12M. To use we have to make 1M solution. For this, take 86mL of stock pure solution and raise up to 1000mL with deionized water.
Caution! Heating may cause explosion, contact with combustible material should be avoided, use carefully as it causes eye irritation. Perchloric acid and 10% Trichloroacetic acid is highly corrosive and handling should be done in a chemical fume hood
Ornithine Decarboxylase Enzyme solution: Stock solution is made as 10mg of enzyme dissolved in 1mL of phosphate buffer (150mM). It means that 0.6 units/mg protein in 1mL
The enzyme is kept at -20ºC and stock solution is prepared in 150mM Phosphate buffer immediately before starting experiment. The enzyme is highly labile and should be kept at 4 ºC while performing the assay.
10mM Picrylsulfonic acid solution: 25.7μL of TNBS solution dissolved in 10mL of 1-Pentanol.
Caution! Store 1-Pentanol in cool place, containers opened must be carefully resealed and kept upright to prevent leakage as it is flammable. It is a harmful by inhalation, cause skin dryness, irritating to respiratory system and eyes. Wear masks and gloves while using it.
- For each sample there must be at least two sets of eppendorf tubes one with the enzyme and another without enzyme. The same case applies to control set also.
- Add the test sample/standard to each set.
- Add 5μL of enzyme from stock solution to one of the sets. The concentration of enzyme is optimized between 10-100μg/mL and ample activity is observed at 50μg/mL.
- Add 400μL of substrate reaction mixture to every eppendorf tubes.
- Incubate for 37ºC for 30 min.
- Terminate the reaction by adding 400μL of perchloric acid (1M) or 400μL of trichloro acetic acid (10%). Precipitation must be done to stop the enzymatic reaction.
- Centrifuge at 5000rpm for 5 min at room temperature.
- Mix 100μL of standards (Ornithine/Putrescine 0.2-100μM) and/or terminated reaction mixture with 200μL of 4N NaOH. At this moment supernatant should be pipetted very carefully and mixing should be done vigorously [Critical Step].
- Add 400μL of 1-pentanol.
- Mix vigourously for 1 min with vortex mixer [Critical Step].
- Centrifuge for 5 min at 2000rpm. Two layers form, bottom hydrophilic and top hydrophobic layer. At this point demarcation line should be clear.
- Transfer 200μL of upper (organic) phase to a fresh tube containing 200µL of sodium borate (0.1M, pH 8.0) and mix. [Critical Step]: pH of Sodium borate is very crucial, it should be 8.0 and mixing should be done properly.
- Add 200μL of TNBS (picryl sulfonic acid, 10mM in 1-pentanol). [Critical Step]: Prepare the TNBS dye reagent immediately before use. It is prepare in 1-Pentanol, so thawing of TNBS should be done beforehand as it is kept at -20◦C. After that mix for 1 min on vortex mixer.
- Add 400μL of DMSO and mix for 1 min. DMSO should be added after proper mixing of supernatant and TNBS.
- Centrifuge for 5 min at 3000 rpm. [Critical Step]: A very fine two layers are observed with top layer contains TNP-Putrescine-TNP, while bottom layer contains TNP-Ornithine-TNP. Sensitivity of the assay depends on the precise pipetting of the upper fine layer. The volume of the layer varies depending on the enzymatic reaction (30-100uL).
- Take upper layer and read absorbance at 426 nm.
- Calculate the percentage of enzyme inhibition using the formula below. For initial screening, we use a threshold of 50% enzyme inhibition as a cut off for compound behaving as ODC inhibitors. However the threshold can be increased or decreased according to inhibitory criteria selected by the investigators. For IC50 determination, plot a concentration responsive graph between compound dose and percent inhibition. IC50 values can be derived using curve fitting methods with statistical analysis.
Control Difference = Mean OD Control with Enzyme-Mean OD Control without Enzyme
Sample Difference = Mean OD Sample with Enzyme-Mean OD Sample without Enzyme
Percent Inhibition = Control Difference- Sample Difference X 100
It is also possible to calculate the amount of product formed by plotting a standard graph of Putrescine and calculating the absorbance of test sample in terms of µM of Putrescine produced.