TITLE: Glutathione peroxidase (GPx) activity assessment for zebrafish brain tissue
AUTHORS: Adrieli Sachett, Matheus Gallas-Lopes, Greicy M M Conterato, Ana Herrmann, Angelo Piato

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Zebrafish are being increasingly used as a model animal in neuroscience research. Here we describe a protocol to assess brain tissue activity of glutathione peroxidase (GPx), an enzyme glutathione family, and act how the main remover of hydrogen peroxide in zebrafish brain and whose main biological role is to protect the organism from oxidative damage.</div></div>

[STEPS]
?. [Preparing the reagents]
The first step is to prepare the reagents to be used in the assessment of glutathione peroxidase activity;
?. [Preparing the reagents]
Potassium phosphate buffer :    1.1.1 Weigh  of monobasic potassium phosphate (KH2PO4) in a beaker of appropriate size;    1.1.2 Dissolve the salt with  of ultrapure water;    1.1.3 Transfer the solution to a  volumetric flask;    1.1.4 Using ultrapure water, complete the solution's volume to reach ;    1.1.5 Weigh  of dibasic potassium phosphate (K2HPO4) in a beaker of appropriate size;    1.1.6 Dissolve the salt with  of ultrapure water;    1.1.7 Transfer the solution to a  volumetric flask;    1.1.8 Mix both solutions slowly in a  beaker following the steps below;Transfer  of the monobasic potassium phosphate (KH2PO4) solution to the beaker;Use a pH sensor to evaluate your solution. Expected conditions:;            If the pH of your solution is lower than 7 adjust the pH adding drops of the dibasic potassium phosphate (K2HPO4) solution;            If the pH of your solution is above 7 adjust the pH adding drops of the monobasic potassium phosphate (KH2PO4) solution;After adjusting the pH of this initial solution, proceed to add, slowly, drops of both buffer solutions (monobasic potassium phosphate and dibasic potassium phosphate). Use Pasteur pipettes to add the solutions. Mix your solutions using a pH sensor, making sure the mix of both buffers is always at ;
6.804 g
90 mL
100 mL
100 mL
8.709 g
90 mL
100 mL
500 mL
50 mL
?. [Preparing the reagents]
EDTA (Ethylenediaminetetraacetic acid)  + Potassium phosphate buffer:    1.2.1 Weigh  of EDTA;    1.2.2 Dissolve completely the EDTA powder in  of the potassium phosphate buffer solution prepared in the last step using a beaker of appropriate size;    1.2.3 Transfer your solution to a  volumetric flask;    1.2.4 Using the potassium phosphate buffer solution prepared in the last step, complete the solution's volume to reach ;    1.2.5 Use a pH sensor to evaluate your solution. Expected conditions:;    1.2.6 Store this solution in an amber flask at ;
0.186 g
90 mL
100 mL
100 mL
8 °C
?. [Preparing the reagents]
Reduced glutathione (GSH) :    1.3.1 Weigh  of GSH;    1.3.2 Dissolve completely the GSH in  of ultrapure water using a container of appropriate size;    1.3.3 Stock this solution at , in samples of  using plastic microtubes;
0.0154 g
5 mL
-20 °C
500
?. [Preparing the reagents]
NADPH (Dihydronicotinamide adenine dinucleotide phosphate tetrasodium salt) :    1.4.1 Weigh  of NADPH;    1.4.2 Dissolve completely the NADPH in  of ultrapure water using a container of appropriate size;    1.4.3 Stock this solution at , in samples of  using plastic microtubes;
0.013 g
10 mL
-20 °C
500
?. [Preparing the reagents]
Sodium azide (NaN3) :    1.5.1 Weigh  of sodium azide;    1.5.2 Dissolve completely the sodium azide powder in  of ultrapure water using a container of appropriate size;    1.5.3 Store the solution at room temperature;
0.0065 g
10 mL
?. [Preparing the reagents]
Glutathione reductase (GR) : this reagent should be prepared on the day of the biochemical assay, it should not be stored for later use;    1.6.1 Each batch of the enzyme has a specific concentration, so every time you open a new flask you should redo the calculations as follows to prepare the solution:Liquid GR:  or  protein:214 U        –  1 mg proteinX              –   1.3 mg proteinX = 278.2 U – 1 mL0.5 U  –     XX =  =  =     1.6.2 Using a micropipette, fill a plastic microtube with  of ultrapure water;    1.6.3 Using a micropipette, collect  of the liquid GR and transfer it to the microtube containing ultrapure water, homogenizing the solution;
278.2
0.001797 mL
0.002 mL
2
998
2
?. [Standardization of H202 solution]
Every 30 days, or whenever a new bottle of hydrogen peroxide is opened, the absorbance of the concentrated solution should be checked.
?. [Standardization of H202 solution]
First, we must calculate the molarity of the H2O2 solution in the bottle:Concentration stated in the bottle:  Molecular weight: 34.0147 g/mol1 M ------------ 1 mol ---------- 1000 mL 1 M ----- -----34.0147 g ------- 1000 mLx -------------- 350 g ---------1000 mL x =
?. [Standardization of H202 solution]
Prepare a  solution () of H2O2:    2.2.1 Follow the calculations below to determine the volume of the primary solution needed:C1 x V1 = C2 x V2 x V1 =  x V1 =     2.2.2 Using a micropipette, collect  of H2O2 and transfer to a  volumetric flask;    2.2.3 Using ultrapure water, complete the solution's volume to reach ;
10 mL
10 mL
9.72
9.72 µl
10 mL
10 mL
?. [Standardization of H202 solution]
Read the absorbance of the solution at  in a microplate reader;
240
?. [Standardization of H202 solution]
Calculate the real concentration of your sample following these steps:A standardized  H2O2 solution has an absorbance of 0.39410 mM H2O2 ---------------- 0.394 Abs                                          X          ---------     Absorbance of your sampleX = Represents the real concentration of the hydrogen peroxide sample
?. [Standardization of H202 solution]
Calculate the real concentration of the bottled solution following these steps:As X (calculated on the last step) Represents the real concentration of the hydrogen peroxide sample10 mM H2O2 ---------------- 10.29 M     X         ---------     ZZ = Real concentration of hydrogen peroxide in the bottle
?. [Preparing the reagents]
Hydrogen peroxide (H2O2) solution: Follow the steps described in section 2 of this protocol to first standardize and then prepare a hydrogen peroxide solution. This reagent should be prepared on the day of the biochemical assay, it should not be stored for later use;
?. [Standardization of H202 solution]
Prepare a  stock solution of H2O2: this reagent should be prepared on the day of the biochemical assay, it should not be stored for later use;    2.6.1 Follow the calculations below to determine the volume of the primary solution needed:C1 x V1 = C2 x V2Z (the real concentration determined on the last step) x V1 =  x V1 = depends on the concentration of your hydrogen peroxide solution    2.6.2 Using a micropipette, collect the determined volume of H2O2 and transfer it to a  volumetric flask;    2.6.3 Using ultrapure water, complete the solution's volume to reach ;    2.6.4 Store this solution in an amber flask at  when needed;
10 mL
10 mL
10 mL
8 °C
?. [Standardization of H202 solution]
Prepare a  solution from your  stock solution:    2.7.1 Follow the calculations below to determine the volume of the stock solution needed:C1 x V1 = C2 x V2  x V1 =  x V1 =     2.6.2 Using a micropipette, collect  of the stock solution and transfer it to a  volumetric flask;    2.6.3 Using ultrapure water, complete the solution's volume to reach ;    2.6.4 Store this solution in an amber flask at  when needed;
10 mL
40
40
10 mL
10 mL
8 °C
?. [Microplate preparation and absorbance reading]
Use a conventional 96-well microplate to run your samples. Tissue sample collection and preparation are described elsewhere;
?. [Microplate preparation and absorbance reading]
You must calculate the sample volume that corresponds to  of proteins. This calculation is based on the Bradford method described elsewhere;    3.1.1 To estimate the volume of the sample corresponding to  of proteins, divide the amount of protein needed () by the total amount of proteins in the sample quantified by the Bradford method (example below);Volume of the sample needed for the assay (µL) = 30 µg / total amount of proteins in the sample µg/µL
30 µg
30 µg
30 µg
?. [Microplate preparation and absorbance reading]
Using an adequate micropipette, fill the wells of your microplate as described below. You should provide duplicates or triplicates of each sample as stated above. Using a micropipette fill the wells in this order: Tissue sample, Potassium phosphate buffer/EDTA, GSH, NADPH, NaN3, and GR. Lastly, you must add the H2O2 solution as quickly as possible using a multichannel pipette while being careful to homogenize the content of the wells. The Potassium phosphate buffer/EDTA volume depends on the volume of the sample. All wells should have a final volume of , so the Potassium phosphate buffer/EDTA is used so that every solution reaches this volume (e.g.,  of the sample +  of the Potassium phosphate buffer/EDTA +  of GSH +  of NADPH +  NaN3 +  of GR +  of H2O2);WellSample (µL)Potassium phosphate buffer/EDTA 0.5 M (µL)GSH 10 mM (µL)NADPH 1.6 mM (µL)NaN3 10 mM (µL)GR 0.5 U/mL (µL)H2O2 (µL)Control01252525252525SamplesDepends on the volume of the sample corresponding to 30 µg  of proteins.Depends on the volume of the sample. Volume needed for the final solution in the tube to reach 250 µL. (125 - sample volume)2525252525
250
50
75
25
25
25
25
25
WellSample (µL)Potassium phosphate buffer/EDTA 0.5 M (µL)GSH 10 mM (µL)NADPH 1.6 mM (µL)NaN3 10 mM (µL)GR 0.5 U/mL (µL)H2O2 (µL)Control01252525252525SamplesDepends on the volume of the sample corresponding to 30 µg  of proteins.Depends on the volume of the sample. Volume needed for the final solution in the tube to reach 250 µL. (125 - sample volume)2525252525
?. [Microplate preparation and absorbance reading]
Set your microplate reader to the following configuration: Kinetic mode with agitation; ; Readings every  for ; ;
340
30 °C
?. [Microplate preparation and absorbance reading]
Place and keep your Potassium phosphate buffer/EDTA   on a water bath at  throughout the analysis;
30 °C
?. [Microplate preparation and absorbance reading]
Read the absorbance of the samples at the set conditions. You should see the absorbance of the sample wells decrease throughout the  of measurement;
?. [Calculating data and determinig results]
Choose the best  interval within the  of sampling. To do so check for the interval where the decrease between absorbances is the steepest;
?. [Calculating data and determinig results]
Calculate the mean absorbance of the wells containing the control solution and the wells containing your samples for each reading. Take into consideration whether you chose to use duplicates or triplicates of your samples.
?. [Calculating data and determinig results]
Calculate the difference between the mean absorbance of the control samples within the  interval that was chosen;Δcontrol = (Mean absorbance at 0 s - Mean absorbance after 60 s)
?. [Calculating data and determinig results]
Calculate the difference between the mean absorbance of each of your samples within the  interval that was chosen;Δsample = (Mean absorbance at 0 s - Mean absorbance at 60 s)
?. [Calculating data and determinig results]
Subtract the Δcontrol value from the Δsample value for each of the samples;Absorbance of the sample (Δ min) = (Δsample - Δcontrol)
?. [Calculating data and determinig results]
Determine GPx activity:GPx activity = (Δ min x final volume (mL) x 1000000 x 1 mg/(6.22 x 1000 mL x  amount of protein (mg))GPx activity = (Δ min x 0,25 mL x 1000000 x 1 mg/(6.22 x 1000 mL x 0,03 mg)Simplifying: GPx activity = (Δ min x 250000)/186.6P.S.: 6.22 mM-1 cm-1 = molar extinction coefficients of NADPH
?. [Calculating data and determinig results]
Final results are expressed as nmol NADPH/min/mg of protein.