Drug Discovery andDevelopment (BHS005-6)Tutorial on GST enzyme assayDr. Prashanth Kumar Bajpe24-02-2021Glutathione-S-Transferase (GST)• Glutathione-S-Transferase (GST) are enzymes which catalyzethe conjugation of Glutathione to a wide range of electrophilicmolecules, therapeutic drugs, carcinogens…..• DetoxificationRole of GSTEnzymatic reaction1-chloro-2, 4-dinitrobenzene (CDNB)ProtocolMaterials neededMaterials & Samples:• Liver homogenates are provided.• 2000µg/ml albumin solution• A 96-well plate• BCA working reagent• 100mM CDNB dissolved in ethanol and stored in microtubes.• 100mM reduced glutathione• Potassium phosphate buffer (100mM, pH 6.5)• MicrocuvettesMeasurement of protein concentrationMeasurement of the protein concentration• Pipette 25μl of each standard or unknown homogenate samplereplicate into a 96-well well• Add 200μl of the BCA working reagent to each well.• Cover plate and incubate at 37°C for 30mins.• Measure the absorbance at 570nm on a plate reader.Measurement of GST Enzymatic activityMake enzyme cocktail:• 980μl of potassium phosphate buffer pH 6.5• 10μl of 100mM CDNB• 10μl of 100mM reduced glutathione• Mix – the solution may be cloudy at first, but should clear up after mixing.• Measurement:• For each homogenate sample and a blank, place 900μl of enzyme cocktail into1.5ml plastic microcuvettes. Incubate at 30°C for 5 mins.• To the blank microcuvette, add 100μl Potassium phosphate buffer to enzymecocktail and zero spectrophotometer.• To the sample microcuvette, add 100μl of sample to enzyme cocktail in themicrocuvettes and mix.• Measure and record absorbance at 340nm for 5 mins (at 0 min and 5 min)Calculation• Plot the standard curve and get its equation.• From its equation, work out the protein concentration for each of homogenate samples andcalculate the• total amount of protein in 100μl sample (you get concentration for ug/ml, however you used 100ul of• homogenate sample for measuring)• Calculate the different in absorbance between 0 and 5 minutes and convert it to per minuteabsorbance• Calculate GST enzymatic activity for each of homogenate samples using the formula below (inU/ml).• GST activity is expressed as nmole DNCB per min per mg protein. (nmole/min/mg)• Calculate how much CDNB is conjugated using the below information• Each unit (U:nmol/min/mg) conjugates 10nmol of CDNB• Information required for data analysis• ➢ The molar extinction of CDNB is 0.0096 μM-1 /cm.Bovine Serum Albumin dilution VialVolume of water (µl)Volume and source ofBSA (µl)Final BSA concentration(µg/ml)A0300 of stock2000B125375 of stock1500C325325 of stock1000D175175 of vial B750E325325 of vial C500F325325 of vial E250G325325 of vial F125H400100 of vial G25I40000 BSA Absorbance Final BSA concentration (ug/ml)Absorbance of BSA at 570nm20001.65215001.24310000.987500.7985000.622500.391250.263250.14600.128 Make the standard curve using the absorbance from the table and calculate the equation tomeasure the concentration of the samples using the standard curveProtein concentration measurement principleThe bicinchoninic acid assay (BCA assay)The colour change from green to purple is proportional to the proteinconcentrationThe assay is not much hindered by detergent in the solutionExample of measurement of GST activityAbsorbance of the homogenate HomogenateAbsorbance at 570 nmReplicate 1Absorbance at 570nm Replicate 2Lamb1.021.1Chicken0.780.85Beef1.381.23 Calculate themean AbsorbanceCalculate theconcentration ofthe homogenateusing theequation in thestandard curveTo measure the GST activityAbsorbance at 0 and 5 minutes ofconjugation HomogenateAbsorbance at 0 minuteAbsorbance at 5 minutesLamb0.1880.871Chicken0.260.799Beef0.0930.393 Calculate theAbsorbance perminuteCalculate thespecific activity ofGST using theequation in thenext pageCalculate how much CDNB is conjugated using the belowinformationEach unit (U:nmol/min/mg) conjugates 10nmol of CDNBEquation to calculate the enzyme activity11Beef: A/min = (1.176 – 0.669)/5 = 0.1014We employ the formula for GST enzymatic activity for each of the given samplesMolar extinction coefficient is an intrinsic property of the chemical or substance whichdetermines how strongly it absorbs light.The molar extinction of CDNB is 0.0096 µM-1 /cmEach unit (U:nmol/min/mg) conjugates 10nmol of CDNBTotal volume: 1 mlSample volume 0.1 mlWhat will you find in the Breo?• Students will be divided into 5 groups• Each group will have a unique data• Assay protocol• Data of sample absorbance-(To calculate the protein concentration)• Data of absorbance of CDNB conjugation, 0 minutes and 5 minutes-(To calculate the specific activity of GST)• Tutorial slides• Tutorial video• Useful literatureAbsorbance values to calculate the standard curve of BSA remains the same for all groupsQuestions?
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