Antioxidant Enzymes - Science topic

Very much interesting question but haven't worked with it.

Agree with Dr Santosh Kumar

Hi Andrès,

I work regularly with the IBR and in general, I calculate it and make the graphs directly with excel.

I'm not sure that there is software available for the calculation.

The different steps are described in the articles. It seems complicated but once the formulas are understood, no worries. You just have to be careful to have all the necessary conditions.

I hope this will help you.

Good luck.

Eric

Dear Raul Diaz Molina

For ABTS radical scavenging assay, 0.1 M potassium phosphate buffer (pH 7.4) is the preferred diluent followed by methanol.

Regards

Thank you for the answers on how to remove haemoglobin in erythrocytes lysates. the answers have brightened my day.

What about electrochemical methods for determination of oxidoreductase enzymes i think they are more sensitive and fast methods

In a optimum condition for growth, I sprayed my plant with cytokinin, the levels of antioxidant enzymes (POD, SOD, catalase) in these plant was higher than control (foliar with distilled water). why?? It's not a results due to reduce free radicals?

Dear Prem,

The above method given by Mehdi Sir is for invivo antioxidant activity in animals but i suggest you the below references for your plant antioxidant estimations...go through them and i hope you find your answer!!!

Determination of SOD, POD, PPO and CAT Enzyme Activities in Rumex obtusifolius L. Annual Research & Review in Biology 11(3): 1-7, 2016; Article no.ARRB.29809

I obtained similar results recently where there was a reduction in the levels of the GSH level but increased activities of GPx, GST, CAT SOD and XO when some drugs were used to treat a disease but all the same, there was increase in lipid peroxidation (MDA level), DNA fragmentation, caspases 3 and 9 activity, mATPase activity anda large amplitude MMPT pore opening both in vitro and in vivo. The curative effect of the drugs with concomitant increase in the antioxidant enzyme activities could not rescue the cells from mitochondrial-mediated apoptosis. Thank you

Greetings Abdulla

The main sources for oxidative stress in cell culture is dimolecular oxygen O2 which necessary for cells to grow in culture this source is more pronounced in serum and proteins free culture media where serum and proteins are a good contributor to antioxidants and keep oxidants/antioxidants status in balance in normal animal cells while this balance is disrupted in cell culture due to removal of serum and proteins as well as addition of some various compounds and regulators known to disrupt such balance such as ethanol.

Every molecule that reacts with free SH groups inhibits GR int that this enzyme bears two SH in the active site. Thus is easy to inhibit its activity but difficult to do it specifically.

To best of my knowledge the best inhibitor is BCNU (carmustine), I have used it and it is very efficient.

Thank you Afyaa, hope you good luck in your research.

No, just make sure they're absolutely clean and handle them with gloves. For fluorescence-based kits use powder-free gloves.

That depends on a couple of questions:

Are you interested in extracellular or intracellular proteins. For extracellular use Na⁺; for intracellular use K⁺. The physiological pH inside cells is somewhat lower (6.8) than that of the extracellular fluid (7.4).

If you want to use ion exchange chromatography, make sure that your buffer ion doesn't bind to the resin. I.e., for cation exchange use an anionic buffer (like HEPES), for anion exchange a cationic (e.g., Tris).

Osmolytes (glycerol, sugar, mannitol) may be useful to adjust the osmotic pressure, but not all proteins require them.

You may need some ions (Ca, Mg) or small molecules (substrates) to stabilise your protein. Don't forget protease inhibitors!

Dear Omar

Add protease inhibitor (prevent proteolysis) and sodium azide (prevent microbial growth) to your extract and store it in fridge 4 degree but not for long time as this may affect enzyme activity.

Hope this helps

I send my papers about this argument.

Regards

Sebastiano

Dear Mohamad

Please find a summary of the Antioxidant Enzymes Assays as an attached pdf file.

Best wishes

Kazem Ghassemi-Golezani

It will also be important to consider the timing of sampling, since your plants might be caught in an oxidative burst with elevated activities, or still/already in a low defense status.

Thanks Helena, I only seen your response now. I have looked around online and found a few kits but when I asked the provider for support the standard answer is that the lysis kit/reagant in question hasn't been tested in combination with the biochemical assay I am trying to run. I will probably end up taking a chance on one of these kits. Thanks for answering :)

That is a great idea, Yuanyuan Liu. Thank you for the suggestion!

Hi Charlotte,

Just saw this old thread but thought I'd respond because I have some relevant unpublished data. I can only speak to the TEAC assay (sometimes called TAS or TAC), described in my 2007 paper "Measuring antioxidants in wild birds." That paper describes the time series of the spectrophotometric readings: a flat low initial absorbance, followed by an increase (initially linear, then plateauing). I recommended not using the commercial method of just taking the reading at 3 minutes because this confounds the timing of the initial increase, and the rate of increase/plateauing. 

What I did not say in that article is that we have good reason to think the initial timing (length of the flat phase) is due to non-enzymatic antioxdants, whereas the rate of increase is likely enzymatic, though we do not have firm proof. All the controls use standard non-enzymatic antioxidants followed by a linear increase (no plateau). In contrast, when we tried to run samples on homogenized chicken tissues, we had no initial delay but a very quick plateau without much linear increase. We got similar results when blood samples had too much hemolysis. My conclusion was that enzymatic antioxidants, which are high within cells but not in plasma/serum, contribute to the plateauing and the slope of the initial linear increase.

This was all 10-12 years ago, so my memory is a bit fuzzy, but I think the interpretation as non-enzymatic is basically right (though with perhaps a few nuances) as long as one uses the method I proposed of using the time to initial increase rather than absorbance at 3 minutes. I would expect similar properties for some of the other assays, but would be cautious in interpretation.

All the best,

Alan

In cool season grasses the complete fertilizer with emphasis on Phosphorus and Potassium is often recommended to improve over wintering. Phosphorus deficiency is most pronounced under cool winter  conditions and is believed related to poor mobilization of P from the soil under that regime. The total antioxidant activity might relate to stress which the plants experience and to the fertility regime. Besides looking at the antioxidant levels it would be prudent to carefully develop an idea of the mineral absorption and look for the interaction of plant performance to these influences. Developing an experimental approach where the levels or nutrients are varied by treatments aimed at the concentration and or frequency of your fertilizer application and then doing both the plant performance, mineral composition and antioxidant assay might give better idea of the inter relationship.  I agree with Ramin's comment that stresses can elicit and induce antioxidant elevation and wish you good luck in your endeavor. Being able to induce the increased antioxidant before stresses are applied can condition better ability to withstand the stress the proposed mechanism is stress is conditioned by tissue oxidation and the higher antioxidant ameliorates the negative consequences on the plant by allowing less damage.  

Please Answer my question...... It will really help me

Well, depends on the level of the oxidative stress (OS). In general OS is adaptive reaction of the body to any alteration of environmental or internal conditions. The first reaction of the AOE to the OS is to increase the activity and/or expression. If OS accelerates, the activity of the AOE may decline due to oxidative damage of the proteins (antioxidant enzymes), or genes responsible for the AOE expression. 

I think it is OK to determine the enzymatic antioxidants in the cotton cotyledons under NaCl and drought stresses. I disagree with Rajesh on using NaCl to induce water deficit since ionic stress will be unavoidable factor. It is better to use PEG polymer of 8000 or more to induce water deficit. PEG 8000 or more decreased the growth medium water potential without entering the cells. It cannot pass through the interstices of the cell wall because PEG is bigger. You have to known this number 8000 is not a real molecular weight.

Greetings

Occurrence of high temperature  concomitant with water deficit stress induces two different  type of defense mechanism in higher plants, namely, induction of anti oxidant enzymes and specific  HSPs in cytoplasm as well as in organelles like  endoplasmic reticulum, mitochondria and chloroplast.  Induction of  antioxidant enzymes are reported  under most type of abiotic stresses including high temperature stress. There is direct relationship exists between antioxidant enzyme activities like SOD, peroxidase, catalase, ascobate peroxidase (in chloroplast), glutathione reductase etc.,  with high temperature tolerance in many plant species. 

Dr. K. Annamalainathan

I found the same as you in rat model of  voluntary a prolonged alcohol intake. It seems to me, that the long during free radicals production damages DNA and proteins, including enzymes. This may result in any events: production of antioxidant enzymes with altered molecular structure, structural damage of normally synthesized enzymes, etc. The cumulative result of all these processes will be decreased enzymatic activity. 

Dear Rafik

Thanks alot for your quick and complete answer, this was very helpful.

Kind regards,

Radia

·         Weighted the organs i.e liver.

·         Added phosphate buffer (pH 7.4) 4 times greater than the weight of organ i.e 1:4.

·         Homogenize it for 15 minutes with the help of pestle and mortar or with the help of homogenizer with short intermissions.

·         Passed the homogenized tissues through muslin cloth to remove the biomass.

·         Filtered the liquid obtained from muslin cloth with the help of whatman filter paper no. 1.

·         Centrifuged the filtrate obtained above step in refrigerator centrifugal machine at 10, 000 rotates per minutes for 15 minutes.

·         Both sediments and supernatants separated for further analysis.

·         All the enzyme isolation steps will be carried out at 4°C.

The supernatants and sediments were stored at 4°C until further analysis.

0.371 g NaH₂PO₄ and 0.270 g Na₂HPO₄ was taken in a flask and added distilled water to make volume upto 100 mL and pH was adjusted 7.0.

I suggest you to follow the classical methods for the determination of the activity of these antioxidant enzymes (see below). All you have to do is to adjust the volumes of the original methods (performed using cuvettes) and keep same concentrations of reagents (although optimisation for your sample is desirable).

Note that:

Your microtiter plate must be optically transparent at the desired wavelength (e.g. 240 nm for H₂O₂/Catalase activity).

The pathlenght in microtiter plates depends on the volume in the each well, in contrast to cuvettes that have a defined pathlength. Make sure to take that into account when calculating the activity, which is done based on molar absorptivity (ε) of substrate or product. The values of  molar absorptivity (ε) are usually given for a pathlength of 1 cm, which often is not the case for microtiter assays. 

References for methods:

Paglia, D.E., Valentine, W.N., 1967. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70, 158–169.

McCord, J.M., Fridovich, I., 1969. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem. 244, 6049–6055.

Aebi, H., 1984. Catalase in vitro. Meth. Enzymol. 105, 121–126.

Paoletti, F., Aldinucci, D., Mocali, A., Caparrini, A., 1986. A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts. Anal. Biochem. 154, 536–541.

Estimation of superoxide dismutase activity:

Superoxide dismutase activity was determined according to Nishikimi et al. (1972).

Reagents:

A- 50 mM phosphate buffer at pH 8.5

B- 1 mM nitroblue tetrazolium

C- 1 mM NADH in the buffer A

D- 0.1 mM phenazine methosulphate

E- Superoxide dismutase (the enzyme extract)

Procedure:

A working reagent was firstly prepared by mixing reagent A, B and C in a ratio of 10: 1: 1 ml. The following solutions were then pipetted:

           Reagent

           Sample

           Blank

Working reagent

             1 ml

             1 ml

E

0.1 ml

-

Distilled water

-

0.1 ml

D

0.1 ml

0.1 ml

The contents of each of the sample and blank were immediately mixed by inversion and the increase in absorbance (rA) at 560 nm was recorded for approximately 5 minutes for both of the sample and blank. The enzyme activity (Unit ml-1) could be calculated as following:

Enzyme activity = [(rA560 blank - rA560 sample) /

rA560 blank] X 100 X 3.75

Hi Liger,

I found the answer.

This answer is from Dr Neha Gupta

Dear Sir,

NBT method for SOD assay is based on principle that NBT undergo photoreduction (which is a blue coloured formazan) on exposure to light by superoxide radicals. It competes with enzyme SOD for superoxide anions. In presence of SOD in reaction mixture, NBT will produce less amount of coloured complex than control.

% inhibition of NBT reduction by SOD = control OD- treatment OD/ control x 100 =X% inhibition.

50% inhibition is equal to 1 unit of enzyme. then X% is equal to 1/50 x X= Y unit.

In your case the SOD calculation will be = (0.389-0.120)/0.389 * 100 =69.15%

50% inhibition =1 Unit of SOD

69.15% inhibition = (1/50)*69.15= 1.38unit

1.38Unit is from =10ul of enzyme extract

Total volume of enzyme extract=1ml=1000ul

(1.38/10 *1000)= 138units of SOD

1ml of extract is from 0.5g of tissue

So SOD units/mg = 138/500= 0.276 units/mg Fresh weight

Hi. The best method for preventing of enzyme denaturing is freezing in liquid nitrogen. by freezing we can protect enzyme.

These are specifications for DTNB

GSSG is soluble in water or phosphate buffer until approx. 0.1 M

J Res Med Sci. 2012 Oct;17(10):938-41.

Comparison of serum trace element levels in patients with or without pre-eclampsia.

Farzin L1, Sajadi F.

In this study, like others, these micronutrients were lower in patients with preeclampsia. However, I don´t now if there is a study that determine these parameters in a predictive model, for example during the first trimester. It could be a good research line.

You have to do 2 techniques- Western and immunohistochemistry. Use antibodies against normal and phosphorylated proteins. Do a literature search to find out at which amino acid/acids, the proteins get phosphorylated. Then look for suitable antibodies. Immuno will tell you the localization of the proteins. If the functions of these proteins are determined by post-translational modifications, then there is no need for doing RT-PCR as this one will tell u about the genes, but will not reflect on end result due to protein functions. Good luck

I think this is a matter of opinion. My preference would be units/mg protein because the liver sample has been processed by homogenization and centrifugation, which amounts to a purification step. Some of the enzyme activity may be in the pellet, so the measurement of the supernatant's activity is not necessarily representative of the liver tissue as a whole.

It doesn't sound too bad, but you never know.  If you're just exploring what happens, it will probably be fine.  You'll learn something from it.  If it's a pivotal experiment for a publication, better re-do it... 

Dear Debaprasad,

I would like to examine SOD, CAT, GPx, POX, NOX enzyme activities in silkworm (digestive system, haemolymph of the larvae). Thank you.

More and more labs are using GSH:GSSG ratios as an indicator of oxidative stress.  Measuring reduced glutathione, GSH, is fairly easy as at contains a free thiol group.  We use Ellman’s reagent (5,5'-dithiobis-2-nitrobenzoic acid (DNTB)) that reacts with GSH resulting in a product that can be measured at 412 nm.

 GSSG is more difficult as it is present at a much lower concentration than GSH and it needs to be reduced in order to be measured. We add a thiol scavenger to our samples to bind with GSH to make a pyridinium salt.  This prevents GSH from binding to DNTB and from oxidizing to GSSG.

 We split each sample into two tubes; one for GSH and one for GSSG.  The GSH sample can be measured with Ellman’s reagent in the traditional way. The GSSG sample needs to have the thiol scavenger added (preferably at the time of collection).  DNTB is then added followed by glutathione reductase to convert GSSG to GSH.  While the scavenger is still present, the reaction rate for DNTB is much faster.  The sample can then be measured at 412 nm.

This link has additional information on the method as well as protocols for cuvette and microplate assays.  http://www.oxfordbiomed.com/gshgssg-assay-1

More and more labs are using GSH:GSSG ratios as an indicator of oxidative stress.  Measuring reduced glutathione, GSH, is fairly easy as at contains a free thiol group.  We use Ellman’s reagent (5,5'-dithiobis-2-nitrobenzoic acid (DNTB)) that reacts with GSH resulting in a product that can be measured at 412 nm.

 GSSG is more difficult as it is present at a much lower concentration than GSH and it needs to be reduced in order to be measured. We add a thiol scavenger to our samples to bind with GSH to make a pyridinium salt.  This prevents GSH from binding to DNTB and from oxidizing to GSSG.

 We split each sample into two tubes; one for GSH and one for GSSG.  The GSH sample can be measured with Ellman’s reagent in the traditional way. The GSSG sample needs to have the thiol scavenger added (preferably at the time of collection).  DNTB is then added followed by glutathione reductase to convert GSSG to GSH.  While the scavenger is still present, the reaction rate for DNTB is much faster.  The sample can then be measured at 412 nm.

 This link has additional information on the method as well as protocols for cuvette and microplate assays.  http://www.oxfordbiomed.com/gshgssg-assay-1

Dear Raei,

GST activity assay by monitoring formation of the GSH-CDNB conjugate at 340nm.

Kindly go through the attached file.

Hope it will help 

Most cytosolic proteins lack disulfide bonds because the environment in the cytosol is reducing due to the presence of reducing agents such as glutathione. To mimic this reducing environment in the extract, a reducing agent is included. This helps keep cysteine (and methionine) residues from oxidizing, which might reduce enzyme activity.

If the enzymes in question are found in an oxidizing compartment, however, (e.g. bacterial periplasm, mitochondria, cell surface), it might be preferable not to include a reducing agent, because the enzyme may have disulfide bonds.

I'm not sure whether a reducing agent in your sample will be a problem for studying antioxidant enzymes specifically. Consult the relevant literature.

In clinical studies, erythrocyte SOD activity with higher values means increased mild oxidative stress and this is an adaptation response .  In the precursors of the mature red blood cell, the radicals increase the expression of the gene for SOD.  

Erythrocyte SOD activity with lower values means increased (severe ) oxidative stress.

Could be the spec as Olkowski indicated. It could also just be nonspecific GS-CDNB reaction since GSH can degrade at temperatures higher than 4 degrees. You will need to subtract the blank as background regardless of how low you can get the blank/nonspecific reaction. What is the percentage of your bank delta abs/min compared to your samples and positive controls? 

It depends on the state of the cell/ tissue. After a mild increment in reactive oxygen species generation, cells are able to increase the antioxidant response and overcome stress. However, after high oxidative damage, cells are no longer able to protect themselves and even antioxidant enzymes could be degraded

I would suggest rather than using recombinant construct one should look at improving the cellular environment by additing potetial antioxidants with previously established use such ascorbate.  Using genetic contruct may be good if one is looking for effect on certain antioxidnat axis but in reality antioxidant defence is a complex process and depends largly on local chemical miliu of cell rather than levels of antioxidant enzymes. 

Dear Arbace,

The attached paper covers the answer to your question.

Namakkal Surappan Rajasekarana, ,

Niranjali S. Devarajb,

Halagowder Devaraj, a,

2.4. Isolation of erythrocytes and erythrocyte membranes

Erythrocytes and their membranes were isolated from control and experimental groups according to the method of Dodge et al. [15] with slight modifications. Packed cells were washed with isotonic saline to remove the buffy coat. Different aliquots of packed cells were thoroughly washed with tris-buffer, 0.31 M, pH 7.4. These were used for the determination of several biochemical parameters. Then the another aliquot of packed cells was subjected to hemolysis by adding hypotonic tris-buffer, 0.015 M, pH 7.2. After 4–6 h, the erythrocyte ghosts were sedimented by high speed centrifugation at 12 000 rpm, for 40–45 min, at 4–6 °C. The supernatant, hemolysate, was used for the analysis of antioxidants. The erythrocyte membrane pellets were suspended in 0.02 M tris-buffer, pH 7.2, and subjected to various biochemical investigations.

2.5. Estimation of protein

The protein content of the erythrocyte membranes was estimated according to the method of Lowry et al. [16].

2.6. Determination of antioxidants

2.6.1. Glutathione estimation

GSH was estimated from the hemolysate after sedimenting the erythrocyte membrane (EM) fraction. An aliquot of lysate was subjected to assay with DTNB and the color produced was read immediately at 412 nm [17].

2.6.2. α-Tocopherol estimation

Erythrocyte membrane content of α-tocopherol was determined after extracting with hexane. The hexane extracts were processed according to the method of Desai [18]using bathophenanthroline, which forms complex with ferrous ions.

2.7. Determination of antioxidant enzymes

2.7.1. Catalase (CAT)

CAT activity of erythrocytes was assayed following the method of Beers and Seizer [19]. The breakdown of hydrogen peroxide on addition of enzyme source was followed by observing the decrease in OD of peroxide solution in ultraviolet (240 nm) region.

2.7.2. Superoxide dismutase (SOD)

The activity of SOD in the hemolysate was determined according to the method of Misra and Fridovich [20]. It is based on the inhibition of epinephrine adrenochrome transition by the enzyme.

2.7.3. Glutathione peroxidase (GPX)

Activity of GPX from hemolysate was assayed according to the procedure of Rotruck et al. [21] with slight modifications. This procedure is based on the reaction between the glutathione remaining in the following reaction with DTNB to form a compound which absorb light maximally at 412 nm.

 

 

2.8. Determination of lipid peroxides by high performance liquid chromatography (HPLC)

Lipid peroxides were measured as malondialdehyde (MDA) products, using the HPLC acetonitrile precipitation assay [22]. Briefly, aliquots of erythrocytes and membranes suspended in buffer were mixed with equal volume of acetonitrile, homogenized and centrifuged to precipitate the proteins. The supernatants were then filtered through millipore membrane (0.2 μm) and 20 μl of samples was injected by Hamilton syringe in the HPLC for detection of MDA. The MDA was identified by comparing the retention time (Rt) with the standards, tetra methoxy propane run under identical conditions. Diene conjugates, indices of LPO initiation, were measured in the lipid extracts of erythrocyte membranes by following the method of Klein [23].

Hoping this will be helpful,

Rafik

 Dear Sel,

Caffeic acid, an extensively studied dietary cinnamic acid derivative, possesses vicinal hydroxyl groups; it undergoes reversible oxidation to initially yield o-quinone, as Pallavi mentioned. This is the initial step in the oxidation of o-diphenols, which can be performed by enzymes, alkaline conditions and oxidizing agents. I presume that by oxidation of caffeic acid (“solution of caffeic acid”) you are referring to autoxidation or oxidation catalyzed by oxidants. Caffeic acid is soluble in organic solvents and hot water. Caffeic acid in certain instances is dissolved using a carrier solution (by dissolving ammonium acetate in water/methanol), and suitably adjusting the pH of the sample solution.

When caffeic acid is oxidized by polyphenol oxidase (tyrosinase) or KMnO4 the first oxidation product formed is the o-quinone of caffeic acid, which is not very stable. Initial oxidation is followed by the generation of caffeic acid oligomers (e.g., tetramers).  Non-enzymic oxidation of caffeic acid occurs by autoxidation and alkaline medium favors this. Autoxidation is dependent on pH. Caffeic acid undergoes reversible oxidation in solutions with a certain pH upper limit. Initial oxidation herein is an o-quinone. After the generation of the quinone, subsequent reactions are the same as those catalyzed by polyphenoloxidase. Typically, a phenol undergoes 1-electron oxidation to generate a semiquinone, which is converted to a quinone. Oxidative coupling (-C-C) of phenols (coupling reaction of the semiquinone radical, an intermediate of one-electron oxidation) is ubiquitous, and generates dimers, which are susceptible to further polymerization. Radical scavenging activity of certain caffeic acid polymers has been assessed. Some reports indicate that these polymeric molecules display much lower radical scavenging potentiality in comparison with that of caffeic acid. 

I left academia in 1997 after having worked in the field of "oxidative stress" for several years. It was already known back then that in vitro antioxidant activity could not be directly correlated with in vivo antioxidant activity. One paper I eventually had published on this subject in 1997 [1] was initially rejected by the editor of another journal on the basis that it "provided only negative results".

Much of what is written on antioxidants is just "hype", whether in the field of cosmetics, nutrition, or medicine. It tells me something about how much background literature research is not done when designing experiments in this field!

Just about anything and everything can be shown to have some kind of antioxidant activity. Water can be very effective at putting out a small fire; so can a plank of wood. These are both antioxidants.  Context matters.

Living systems are essentially reducing environments in which oxidative processes are allowed to occur under carefully controlled conditions. That control is exerted by various antioxidant molecules and systems. These oxidative processes release energy from fuel molecules. Without antioxidant defences, we would ultimately self-combust. Water plays a significant part in our antioxidant defences. So do free-radical scavenging antioxidants; so do other classes of antioxidant molecules. But we have to understand that antioxidant chemistry is just chemistry. It was demonstrated many years ago that when a water soluble radical-initiator is added to whole blood, endogenous antioxidants are consumed sequentially: this is driven by the oxidative / reductive electrochemical properties of the reactants. Ascorbic acid is consumed most rapidly, followed by bilirubin then uric acid then plasma vitamin E [2].

Ascorbic acid acts as a sacrificial antioxidant. In taking a hit from a potentially-damaging oxidant free radical species, it traps the unpaired electron to form a particularly benign free radical species, namely semidehydroascorbic acid. This can take a second hit to become dehydro(ascorbic acid), the original ascorbic acid molecule having quenched two unpaired electrons in the process. But perhaps most importantly, the dehydro(ascorbic acid) can then be metabolically reactivated back to ascorbic acid at the expense of glutathione. The glutathione, in turn can be metabolically reactivated at the expense of NADPH. And NADPH is produced in mitochondria, our intracellular "batteries" [3].

Innumerable examples of substances with ascorbic-acid-like antioxidant activity in vitro can be found in the literature. But ascorbic acid is probably unique in the fact that when it quenches a potentially damaging free-radical, it does not itself become a new potentially-damaging species. Ultimately, this is why results from in vitro antioxidant studies are so difficult to interpret. In silico predictions are only as good as the data used to develop the in silico model.  "Garbage in; garbage out".

So, we humans [probably] just need to ensure we have an adequate supply of ascorbic acid / vitamin C in our diet. And, more importantly, that our diets do not add to the background oxidative stress we all inevitably have to endure as the price we pay for extracting energy from the food we eat. Adding random new antioxidant wonder-molecules to our diet is probably a bad thing to do unless these molecules are first shown to produce chemically and physiologically benign products on trapping reactive oxygen and reactive nitrogen species.

1. http://dx.doi.org/10.1016/S0378-8741(97)01510-9

2. Niki E. Antioxidant compounds. In: Oxidative Damage and Repair (Ed. KJA Davies). Oxford: Pergamon Press, 1991, pp 57-64.

3. Schmidt RJ. Redox homeostasis and microbial colonisation of wounds: new insights into the energy economy of chronic wounds, Journal für Anästhesie und Intensivbehandlung 3(3): 26-31 (1996) [http://www.pabst-publishers.de/Medizin/med%20Zeitschriften/jai/1996-3/art-6.html ; downloadable from http://bit.ly/VQBqm4]

For visualizing bands with superoxide dismutase, catalase or glutathione peroxidase activity you don't need a fluorescence lamp.

Please refer to the attached publication for more information.

Cheers, Christian

Dear Ifeoluwa T Oyeyemi

I am attaching literature on the anticancer properties. I hope they will be useful to you.

I recommend the fruit of Rhus coriaria L. (Anacardiaceae) for your research.

Good luck.

Dear Prabakaran,

Majority of enzymes extracted in buffer will lose their activity, when stored at 4^oC for over 12 to 24 h. If you want to store for longer time you can freeze and store them in freezer (may be at 20^oC). However, please ensure that the samples are not subjected to repeated freezing & thawing (this is often experienced due to power failure, which many of us might overlook). Whenever you wish to measure enzyme activity, you can take out the sample, thaw it & measure activity(activities) of whatever enzyme(s) you are interested in. Please do not refreeze or store the sample(s), ones you thaw it(them).

Thanks for your message

Yes! But this is not a measure of antioxidant activity, but a marker of the accumulated oxidative stress/damage.

Relative high contentes of sugars, phenols and proteins might interfere the reading of the adduct at 532nm. Try Hodges modification of the method that consideres interfering compounds.

Hodges, D.M. et al., 1999. Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta, 207(4), pp.604–611.

[Edited to note that is a mesure of oxidative stress/damage on the lipidic phase]

We used RANSOD SOD kit from Randox Company. We adapted to 96 well microplate and results were consistent and reproducible. With this setting, the kit gave us 80  tests per 20 ml reagent  bottle.

Redox regulation is a complex field.  Please, find attached recent reviews. Dr wong is right!

Correcting for (significant) control responses always poses a risk. If the respons is real and the experiment was conducted properly, you should assume that your exposed organisms were also exposed to the stressor that invoked the control respons. Hence your exposures are actually exposed to a mixture of stressors, in your case diesel contamianted sediment and the unknown stressor. There is no way of knowing if the mixture is additive (which you assume correcting for the control respons) or follows another mixture model, e.g. potentiation, inhibition, ...

Yes, definitely there is. The reducing equivalents from NADPH enter the glutathione system either with the help of the FAD-dependent enzyme glutathione reductase (GR) or the thioredoxin reductase/thioredoxin couple (TrxR/Trx). The electrons are subsequently transferred to endogenous electrophilic compound such as glutathione disulfide (GSSG), yielding two molecules of reduced glutathione, GSH. The disulfide-reducing GR and TrxR could act as electron relays to tap into the NADPH pool, GSH is a versatile adapter molecule, and the glutathione system serves in most aerobic cells and organisms as the central metabolic network to remove or modify electrophilic compounds inherently present inside (endogenous) or outside (exogenous) the cell. Please read more on glutaredoxin systems (based in one reference that I had used the ff are on glutaredoxin systems - Methods Enzymol., 252 (1995), pp. 283–292; Biochim. Biophys. Acta, 1780 (2008), pp. 1304–1317; and Biochemistry, 48 (2009), pp. 1410–1423.)

Nanoparticles act as signaling molecules for conversion of complex carbohydrates, lipids and proteins to simpler metabolites which breakdown easily and release some ATP to be utilised for seed germination.

you will extract these enzymes in 50 mM phosphate buffer containing 1.15 KCl

SOD can be measured by markuland and markulangd for example

Pomace is rich in lot of polyphenols, for example quercetin is powerful and very common in apple.

Dear Tanjina,

Do you think is a suitable method with ethanol and chloroform?Could be apply to others protocols?thank you

It is obvious that the problem makes bubbles of oxygen generated in reaction hydrogen peroxide and catalase. Try to decrease the concentration of H2O2 or the concentration of the sample.

 If there is no result, maybe the attached procedure could help to solve the problem.  

you might want to search vitamin E .there was a study published in the 1970s or 1980s in Germany using emulsified vitamin E..the paper was a pig to market study and showed a reduction in pig deaths in those that consumed emulsified vitamin E. the study was reported by Mucos pharmaceuticals from Germany

I have attached the protocol for APX isozyme detection.

Apart from this for CAT you can follow the ferricyanide method suggested by Woodbury et al. (1971). For SOD, the method suggested by Beauchamp and Fridovich (1971).

Dear Abdullah I attach a chapter from my PhD thesis I hope it will be benefit to you

Superoxide dismutase catalyzes the dismutation of the very active oxygen species  superoxide anion into molecular oxygen and the less harmful hydrogen peroxide, and this reaction provides a very important defense against oxidative damage.  The most commonly used method to measure SOD activity is by the inhibition a reaction dependent on superoxide anion where superoxide ions are to be generated first, say from the reaction between xanthine and molecular O2 in the presence of catalyst xanthine oxidase to form two more products in addition to the superoxide anion and these are uric acid and hydrogen peroxide. Then another reaction is to be initiated say a substrate that easily gets reduced after the reaction with the newly formed superoxide anion, like tetrazolium salt (nitroblue tetrazolium, NBT) that is being converted into a formazan dye (NBT-diformazan), the rate of this second reaction can easily be monitored using a spectrophotometer that measures the amount of the colored NBT-diformazan dye product. Addition of SOD into this reaction therefore reduces superoxide ion levels, thereby lowering the rate of formazan dye formation. SOD activity in the experimental sample is measured as the percent inhibition of the rate of formazan dye formation. In this case there a few blanks to prepare--sample blank (no SOD), reagent blank (no enzyme solution for the first reaction generating superoxide ions, no xanthine oxidase), and the combination of sample and reagent blank. The blank that does not have the SOD extract sample is expected to have higher absorbance since more colored formazan dye will be produced. The one with SOD extract sample will inhibit the formation of formazan dye product and a 50% inhibition is equivalent to 1 unit of SOD. You may search the Internet for more related work.

Apart from their role in countering ROS, they could also have a role in inhibition of polyphenol oxidation and the associated production of quinones which are inhibitors of plant growth and have been linked to tissue necrosis in plant tissue culture.

see 1. http://www.academicjournals.org/article/article1380127753_Cheniany%20et%20al.pdf

and 

2. http://scholar.google.com/scholar_url?url=http://www.researchgate.net/profile/Loic_Lepiniec/publication/6637454_Flavonoid_oxidation_in_plants_from_biochemical_properties_to_physiological_functions/links/0912f50f7122ba3aa3000000.pdf&hl=en&sa=X&scisig=AAGBfm06zMrxz32KEJFZy5MJzPzTCqtH9A&nossl=1&oi=scholarr

Hello,

your question is not clear.

In fact you do not need total protein for enzyme measurement. You can assay the enzyme activity without knowing total protein concentration. But, you need to measure TP because it is used to express the activity of enzymes.

μmol/mg protein..

see one of my publications (uploaded):

"Physiological effects of nanoparticulate ZnO in green peas (Pisum sativum L.) cultivated in soil"

Dear Mohammed

You can see below a method for measuring catalase (CAT) activity:

The plant sample is ground at 4 oC in a porcelain mortar with homogenization buffer containing 100 mM K2HPO4 pH 7.0, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and 0.3 % ethanol. The homogenization is carried out using 5 ml buffer per g fresh sample weight. The homogenate is centrifuged at 3,300 g for 5 min, at 4 oC. The resulting supernatant is subsequently used for both CAT activity and protein concentration estimation. CAT is assayed by its depletion of H2O2, followed at 240 nm (Blum and Fridovich, 1983).

The CAT assay consists of mixing (at 30 oC) 0.9 ml sample (proper dilution of the homogenate supernatant) and 0.1 ml 0.09 M H2O2 stock solution (made fresh in homogenization buffer). The linear absorbance decrease (vs time) of the assay mixture is measured at 240 nm in a spectrophotometer. The absorbance decrease rate of consumed H2O2 is converted to CAT units (U) from a pure CAT standard curve.

For catalase activity as per Anderson et al. (1995) -

Let us presume that you observe decrease in OD (Absorbance) at 240 nm by 0.9 (i.e. A = 0.9) in one minute.

Molar extinction coefficient for H2O2 at 240 nm is 36 M-1cm-1

Path length is 1 cm

Then concentration of H2O2 consumed (in M) = 0.9/36 = 9/360 = 1/40 = 0.025 M

In other words 0.025 M H2O2 was consumed. 0.025 M = 25 mM

25 mM means 25 micromoles/ml

In case if total volume of reaction mixture in cuvette is one ml you can use this value directly (if it is 2 ml then mutiply it by 2 and if it is 2.5 ml multiply 25 micromoles/ml with 2.5).

Let us now suppose that you had taken 100 microliters (i.e. 0.1 ml) enzyme extract (may be crude enzyme extract) and it contains 20 micrograms of protein.

In other words - catalase in 20 micrograms (i.e. 0.02 mg) of protein has potential to catalyze breakdown of  25 micromoles of H2O2 in one minute.

Catalase activity/mg protein/min = 25/0.02 = 2500/2 = 1250

You can calculate peroxidase activity in a similar manner using extinction coefficient for tetraguaicol.

If you wish you may visit our lab and learn protocols along with calculations with me, my doctoral and post-doctoral students.

perform the assay in 1.5 ml tubes and finally read in 96 well plate , assay require a boiling step and u may not use the plate.. However alternative assays such as isoprostanes estimation which also indicate the lipid modifications can be done in standard 96 well plates using ELISA. attached here is working protocol for mda assay i routinely use.  Do not use perfused of fixed brain slices (fixed in formaldehyde) it will not give MDA assay 

Best 

Hi Bostjan,

For determination of Antioxidant enzyme activity or oxidation product you need 50,000 to 500,000 Cells. For determination enzyme activity, as you know, we encounter to signal amplification and thus we need lower cell number (10,000 to 100,000, mean 50,000). But for determination of oxidation products such as MDA, you have not amplification and can determine MDA directly based on TBA reaction, so you need more cell number (100,000 to 1000,000 mean 500,000 cells).

For solid tissue you need average 100 mg tissue for preparation a homogenate 100 mg/ml.

After homogenization, centrifuge and separation supernatant, you can determine antioxidant enzyme activity or oxidation products.

best

mehdi

     

I dilute sample two time first: when extract by (1) 25 mM Phosphate buffer and second: in(2) 50 mM Phosphate buffer contained 0.25 mM ascorbic acid and 10 mM H2O2 .

if I will dilute third time in 1 or 2 buffer up

I used H2O2 concentration 3%

and what polarographic method did you have assay

sent to me please