BHS005-6 (Drug Discovery & Development) | My Assignment Tutor

Practical threeBHS005-6 (Drug Discovery & Development)Enzymatic activity assay for Glutathione S-TransferaseIntroductionGlutathione S-Transferase (GST) is a Phase II metabolic enzyme involved indetoxification of a wide range of chemicals. Identification of GST is performed bywestern blotting or more easily by enzymatic activity assay.Enzyme Reaction: Glutathione –SH + CDNB → Glutathione –S-CDNBThe reaction is measured by observing the conjugation of 1-chloro, 2,4-dinitrobenzene (CDNB) with reduced glutathione. This is done by watching anincrease in absorbance at 340nm for 5 mins. One unit of enzyme will conjugate10nmol of CDNB with reduced glutathione per minute at 25°C.Materials & Samples:Liver homogenates (pig, lamb and chicken) are provided.2000µg/ml albumin solutionA 96-well plateBCA working reagent100mM CDNB dissolved in ethanol and stored in microtubes.100mM reduced glutathionePotassium phosphate buffer (100mM, pH 6.5)MicrocuvettesMethods1. Measurement of the protein concentration in the homogenate samplesby BCA assaya) Preparation of diluted albumin (BSA) standards as guided by the tablebelow: VialVolume of water(µl)Volume andsource of BSA(µl)Final BSAconcentration(µg/ml)A0300 of stock2000B125375 of stock1500 C325325 of stock1000D175175 of vial B750E325325 of vial C500F325325 of vial E250G325325 of vial F125H400100 of vial G25I40000 b) Measurement of the procedure:Pipette 25μl of each standard or unknown homogenate sample replicate intoa 96-well wellAdd 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.2. Measurement of the GST enzymatic activity1) Make enzyme cocktail:980μl of potassium phosphate buffer pH 6.510μl of 100mM CDNB10μl of 100mM reduced glutathioneMix – the solution may be cloudy at first, but should clear up after mixing.2) Measurement:For each homogenate sample and a blank, place 900μl of enzyme cocktailinto 1.5ml plastic microcuvettes. Incubate at 30°C for 5 mins.To the blank microcuvette, add 100μl Potassium phosphate buffer toenzyme cocktail and zero spectrophotometer.To the sample microcuvette, add 100μl of sample to enzyme cocktail inthe microcuvettes and mix.Measure and record absorbance at 340nm for 5 mins (at 0 min and 5 min)Calculations:1. Plot the standard curve and get its equation.2. From its equation, work out the protein concentration for each of homogenatesamples and calculate thetotal amount of protein in 100μl sample (you get concentration for ug/ml,however you used 100ul ofhomogenate sample for measuring)3. Calculate the different in absorbance between 0 and 5 minutes and convert itto per minute absorbance4. Calculate GST enzymatic activity for each of homogenate samples using theformula below (in U/ml).5. GST activity is expressed as nmole DNCB per min per mg protein.(nmole/min/mg)6. Calculate how much CDNB is conjugated using the below informationEach unit (U:nmol/min/mg) conjugates 10nmol of CDNBInformation required for data analysis➢ The molar extinction of CDNB is 0.0096 μM-1 /cm.


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