Questions related to Antioxidant Enzymes
I am trying to measure GUS expression, and was wondering if anyone knew what concentration hydrogen peroxide inhibits the GUS protein???
Thanks ! Any help is appreciated!
I will be thankful if anybody answer my question and send me a clear protocol to measure ABTS Radical scavenging activity of protein samples.I want to measure antioxidant capacity of my samples by ABTS assay.
Hi. I'm working with soil enzymes and I have an issue because i saw several papers that used "integrated biological response" or IBRv2 to integrate data from enzymes and build a hexagonal star plots (based on this paper from Beliaeff and Burgeot "Integrated biomarker response: A useful tool for ecological risk assessment"
See some examples:
Is a software (R package? specifical software? excel file?) available to calculate this index and build the hexagonal star plots? Or is only to calculate the data and after this, make the hexagonal star plot (radar chart) in SPSS?
I know the "Biomarker Integration Data Expert System", but this system is more appropriated for worm enzymes than soil enzymes.
Thank you in advance!!!
Haemoglobin is a serious problem during purification and analysis of antioxidant enzymes from red blood cells. For this reason it could be necessary to remove Hb in order to obtain a clear supernatant.
Good day all
Can someone guide me on the references of methods used to measure enzymes or proteins newly discovered in the blood serum?
What are the main references (book or research) for one or more, that specify methods for measuring newly discovered enzymes or proteins, in which simple manual methods are used.
Thanks in advance
The systemic antioxidant therapy is one of the therapeutic options for oral mucosal lesions. In Localized delivery of antioxidant molecules to the oral mucosal lesion, will the oral mucosal cells uptake/absorb the antioxidant molecules? Will the antioxidant molecules perform their action when delivered locally? Which route of delivery (systemic/local) of antioxidant molecules can have better activity?
The presence of free radicals in photosynthetic and respiratory systems is inevitable, even in optimal condition for growth.
In these conditions, How much free radicals can be loss in yield?
I want to do estimate antioxidant enzymes activity of crude protein extract obtained from salt stress induced cotton seedlings. Can anyone give me standard protocols for SOD, ascorbate peroxidase, catalase and other antioxidant chemicals? Thanks in advance.
In a research study, I have observed induction of apoptosis (DNA fragmentation, Caspase activation and PS externalization) by herbal extract on cancer cell lines. Here I would be able to find out the TNFR1 receptor mediated apoptosis with also activation of p53. Further I estimated antioxidant enzymes levels and found elevation in the activities of superoxide dismutase, peroxidase and catalase with also inhibition in the total RONS levels.
Now, I am not able to justify the elevation in the antioxidant enzymes activities with induction of apoptosis. If somebody reply, it would be great help. Thank you in advance.
I know that cells under tissue culture conditions are different from those in vivo in many ways, but what exactly could trigger antioxidant enzymes such as POX, SOD and CAT in tissue culture media?
I want to perform an inhibition study of GR in fish primary hepatocytes. Is there any Glutathione reductase specific inhibitor which can inhibit only GR not other disulfide oxidoreductase? Thanks in advance.
I have plant treated with different concentrations of copper. Interestingly, the callus exhibited increased levels of MDA, despite enhancement in their biomass. There was stimulation in some antioxidant enzymes such as SOD, POD and GST. How can you explain this? Have you faced this before?
Most paper that discuss heavy metal mediated-oxidative stress, particularly copper as a transition metal, relate the inhibition in growth or biomass to the increase in lipid peroxidation concomitant with inhibition or even stimulation in the antioxidant defense system, in contrast to my results.
I know that among the strategies the plant may employ is to repair the degraded membranes and refold the unfolded or misfolded proteins, but I am not sure if that is the situation for my samples. If it is, should it appear as decrease or increase in lipid peroxidation?
I want to quantify protein content as well as some antioxidant enzymes. I want to prepare a crude extract which can be stored because i would not be able to quantify all enzymes at a time.
I was thinking to grind in liquid nitrogen, then use phosphate buffer, centrifuge and use the supernatant as the crude (and add some glycerol for storage).
I know that there exists a direct relationship between lipid peroxidation and SOD, Cat, GPX etc., But there is lot of ambiguity when it comes to working with a particular cell line. Please post your inputs here. Thank you.
Assalamualaikum and Good day. Does anyone here working on antioxidant enzymes such as SOD, CAT, APX, DHAR, MDHAR, GR, GPX? I desperately need a full protocol or working procedure for the above-mentioned enzymes assay (I mean the step-by-step protocol). I did refer to many journals but failed to get the full procedure. I would appreciate if any of you here can share it with me or suggest the best website to retrieve the protocol. Thanks in advance.
Which antioxidant enzymes could give us more information about the stress level on the plant: APX, GR, SOD or CAT (or any combination)?
For example: APX and GR, or APX, SOD, and GR, etc.
Can someone suggest a lysis reagent/protocol suitable for 96-well yeast lysis which is compatible with protein assay kits?
I want to assay the activity of antioxidant enzymes in the yeast Candida albicans using commercially available assay kits. I would like to lyse the yeast cells in 96-well plate format and am looking for a simple way to do this. I cannot use beads as I do not have the equipment. I have been looking around at various lysis reagents online but many of them have detergents - the kits I want to use to assay enzymatic activity have not been tested in combination with detergents so I would ideally like to gently lyse my cells without detergent. Is this even possible?
Thanks in advance,
Karen O'Hanlon Cohrt PhD
Hi, I will be studying the effects of organic arsenic exposure on rat's long bone for my master's degree and I am planning to use the right femur to measure the lipid peroxidation status (malondialdehyde level) and antioxidant enzymes (glutathione peroxidase and superoxide dismutase). I have found a method that requires the femur to be snap frozen at -80 degree Celcius and homogenised by grinding using pestle and mortar. I am a little bit unsure about the part where MDA and oxidative enzymes are measure. Would anyone be able to tell me the optimal method for doing these tests? I was also wondering if there were any other methods of obtaining the bone homogenates? You are more than welcome to share your research experience here. Thank you!
In most study measuring plasma total antioxidant capacity (using different assays within and between studies), the authors assume it describe the activity of non-enzymatic antioxidants only, usually without any justification.
In a recent paper, this was justified by the supposedly low activity of antioxidant enzymes in the plasma. But then, there would be no point to all the studies measuring enzymatic activity in the plasma, and sometimes finding meaningful biological effects.
In a much older study, when one sample was treated with an inhibitor of the superoxide dismutase (NJ-diethyldithiocarbamate), there was "no effect" (N = 1) on TAC measured by the TRAP method (total radical trapping antioxidant parameter). And I am not sure whether the radical trapping activity of diethyldithiocarbamate itself could not interfere, here.
I did not find other studies properly examining the relative contribution of various antioxidant, and especially major enzymatic and non-enzymatic antioxidants, to the total antioxidant capacity measured through different assays, although there has been a lot of interest in evaluating contributions of individual compounds (e.g. uric acid), but often correlatively, without correcting for potential correlations or interactions between antioxidants.
Does anyone knows about such studies, to back the claim that total antioxidant capacity is mostly non-enzymatic?
My experiment is on cold tolerance of winter season grasses in field condition, and different NPK fertilizer levels are applied to optimize dose for coping winter stress.
Now i want to know Antioxidant enzymes activity will be higher in lower levels of NPK (where plants are very poor due to cold) or in higher levels of NPK (where plants are healthy)......
I am doing experiment on cold tolerance of winter season grasses in field condition, and different NPK fertilizer levels are applied to optimize dose for coping winter tress for northern clime of china.
Now i want to know Antioxidant enzymes activity will higher in lower levels of NPK (where plants are very poor due to cold) or higher levels of NPK (where plants are healthy)......
I have found many in literature data. Some authors describe increased levels of non-enzymatic antioxidant & enzymatic antioxidant (SOD, CAT, GSH, GST etc) under abiotic stress(Salt,heavy metal etc) for plants while others reported inverse correlation.
What are the factors influencing those results?
Do antioxidant enzymes increase or decrease under oxidative stress during time period like after 1 week level increased but 2nd week decrease 3rd week again activated ?
Is production of non-enzymatic antioxidant & enzymatic antioxidant are unpredictable?
If any literature supported above line please share link/paper.
Is it possible for cotton seedlings (10 days old) to produce antioxidant enzymes in cotyledons and other parts under short duration (6h,12h and 24h) of salt and drought stress (stress treatment in distilled water supplemented with various concentrations of NaCl and PEG)? I came across so many articles dealing with leaf and other plant parts only not with cotyledons. I want to estimate antioxidants in whole seedlings after stress tretament. Kindly give your suggestions. Thanks in advance.
There are many studies about oxidative stress induced by EtOH and the role of antioxidants and non enzymatic antioxidants in EtOH detoxification and its metabolites. I have found evidence for decreasing the activity of these enzymes along with a strong increase in oxidizing species.
Needles were grinded in liquid nitrogen and homogenized in phosphate buffer but some authors used sonication, dessication or filtration before enzymatic analysis... what is the best method to prepare needles without degrade antioxidant enzyme (catalase, superoxide dismutase, ascorbate peroxidase)
iam going to quantify the total protein and total lipids in fish muscles and antioxidant enzymes in liver , so i want to know what is the best buffer protocol for homogenization of these tissues?
Can any one suggest me the protocol for estimation of antioxidant enzymes (SOD, Catalase, Peroxidase) by micro plate reader and its calculation?
I have the Abs. values and i want to convert it to mg/g FW or any other unit, the reaction mix was riboflavin, L-methionine and NBT.
I measured activity of SOD enzyme with NBT method by Spectrophotometer. SOD (EC 22.214.171.124) activity was determined by measuring its ability to inhibit the photoreduction of nitro bluetetrazolium (NBT) according to the methods of Beauchamp and Fridovich (1971).
I want to know how i can transform the initial absorbance to Unit/g.fw.
Please explain step by step method for learning this subject.
The information that I have obtained from Spectrophotometer are the following:
The absorbance of Control at 560 nm= 0.389.
The absorbance of Sample at 560 nm = 0.120.
Total volume buffer extraction=1mL.
Total reaction volume for read the absorption= 1mL.
Leave sample for extraction=0.5 gr.
I want to carry my plant tissue abroad and it is not possible to maintain the samples in -80 centigrade so I need to freeze dry and carry samples. Will it affect the antioxidant enzyme activity?
Could you please suggest me what can I use to dissolve these chemicals in water to make molar solution?
The pathophysiology of preeclampsia is not clearly understood. Several studies point to the inadequacy of blood supplied to the placenta. This causes the placenta to release chemical agents. These chemical agents cause damage to the endothelium of blood vessels, alteration in metabolism and inflammation (Adu-Bonsaffoh, et al., 2015; Roberts et al., 2003). Oxidative stress is also thought to play a role. Reduced perfusion of the placenta generates free radicals in the maternal environment (Anto, 2015; Howlader, et al., 2009; Hubel, 1999; Roberts et al., 2003). Antioxidant enzymes Superoxide dismutase and Glutathione peroxidase play roles in mopping up these free radicals (Akinloye et al., 2010).
Micronutrients selenium, manganese, copper and zinc are important nutritional antioxidants that serve as cofactors of the enzyme Superoxide dismutase. Selenium is a cofactor of Glutathione peroxidase (Xu, Shatenstein, & Luo, 2009; Sayyed & Sontakke, 2013; Salles, Galvao, et al., 2012; Rossi & Mullin, 2011; Roberts et al., 2010; Thomas & Shenoy, 2014; Akhtar, Ali, Begum, & Ferdousi, 2013; Al-Jameil et al., 2014 ;Endeshaw, Ambaw, & Aragaw, 2014).
Is there any clinical/research evidence that supports this hypothesis that low levels of these micronutrients are implicated in preelcampsia? Thank you.
I urgently need to determine the previous parameters and I ask for the most convenient method for determination of each other.
Are the previous antioxidant enzymes have post-transitional modification and must determined by western blotting techniques or can be determined by RT-PCR?
Are the previous antioxidant enzymes can be determined by convenient coloremetric method?
In some article, the enzyme activity has shown in terms of gm wet wt but, I used to measure in terms of mg of protein. Is there any difference (technical, bio-chemical) between these?
By mistake, I have kept dissected out tissues in zero degree overnight. Should I proceed with it for antioxidant enzyme assay?Thanks in advance.
can any one help me how to measure the antioxidant enzyme activities in insects especially in silkworm larvae?
tissues include: liver, muscles and ovary
biological fluids: serum and whole blood
hen eggs and sperm
I want to assay the enzyme activity of different antioxidant enzymes. Should I use any reducing agent in homogenizing buffer? I have gone through some protocols for assay and the reducing agents like DTT, β-mercaptoethanol have used in the homogenizing buffer. I am a bit confused about the use of this reducing agent in enzyme assay. How the three dimensional structure of enzymes is maintained in the presence of reducing agent? What is the function of reducing agent in homogenizing buffer in the context of enzyme assay?
Dear experts, I am a bit confused with the expression pattern of antioxidant enzymes i.e CAT, SOD and GST. I am checking oxidative damage potentiality of certain nanoparticle on primary hepatocytes. I used to treat cells upto 24 hrs. In the early stages of treatment I am getting down-regulation of these enzymes in their mRNA and protein level. Is this down-regulation an indication of starting stress? If it is, then what is the explanation? Kindly help me. Thanks in advance.
To assay the GST activity from liver lysate, I have used 0.5 mM GSH, 0.5 mM CDNB and 10 µl of tissue supernatant. I have assayed with the increase in the absorbance at 340 nm. I have set blank with 0.1 M potassium phosphate buffer with 1 mM EDTA . When I have added only GSH and CDNB (without tissue supernatant) there was an increase in the absorbance, which indicates the formation of GSH-CDNB adduct automatically. So, should I deduct the rate of GSH-CDNB adduct formation from the rate of enzymatically formed GSH-CDNB adduct? What should be the calculation to get the actual GST activity?
I have found many disparencies in literature data. Some authors decribe increased levels of antioxidant enzymes (SOD, CAT, GSH, etc) under diabetes or inflammation, while others reported inverse correlation.
What are the factors influencing those results?
Is it a question of physiopathological stage?
Does anyone know if there are specific overexpressing mouse lines or over-expressing viral constructs for increasing endogenous antioxidant activity in a specific type or cell or tissue type? I am specifically interested in changing antioxidant levels in brain-- does anyone know of a transgenic mouse line with this ability, a virus that might overexpress something like SOD2 or Glutathione or a protocol for injecting intraventriculalry specific molecules that increase antioxidant activity?
There are a lot of papers where it is well reported how measure activity enzymes in red blood cells.In fact, most part of them show very famous protocol such as Carlberg I (methods enzymology 1985;113:484-90). However some papers don't show the purification steps from red blood cells before analysis activity.Even, Several authors report a cromatography purification step.
A fast and suitable purification method is recommended.
Solution of caffeic acid and tannic acid (each) was oxidized so converted to quinones. (may be benzoquinone)
I'm guessing the quinones formed oligomers with each other and wondering if they still have radical scavenging power.
Some references mention that they will lost the anti-oxidant capacity, but some say they won't.....
Recently, I have attended two conferences in Europe (EuroFedLipid, Florence, Italy and EuroFoodChem, Madrid, Spain). During many presentations on ANTIOXIDANTS, I have observed that, many researchers have repeatedly highlighted in vitro antioxidant studies are really useless and it does not have any 'real' significance! They have argued that the results will never have any significant correlation with in vivo studies.
We can find thousands of studies on in vitro antioxidant assays of plant extracts, synthetic compounds, nanoparticles, so on. I am sure there are so many researchers are working on it and writing their papers.
What is your opinion? We must stop such assays and spend our time and energy on other aspects of science?
I am investigating the hepatoprotective effect of some medicinal plant on chronic carbon tetrachloride induced liver injury. I observed that although some of the medicinal plants increased the antioxidant enzyme (SOD, CAT and GSH) level in rats co-exposed to CCl4 and the extracts while they also increased lipid peroxidation and markers of hepatic injury such as ALT and AST.Has anyone experienced something similar? If yes, what are the likely reasons why this happened?
i couldn't find the incubation temprature of extraction of plant cells to determine activity of SOD and APX antioxidant enzymes. could you help me, please?
is that the high content of sugar in the extract may act in the TBARS assay? because i tried to evaluate the antioxidant activity of date fruit using this assay but it doesn't work.
I want to determine SOD activity in liver, gill and muscle and in many publication the method use "xanthine method" or "pyrogallol method"
I exposed Jurkat E6-1 cell to plant secondary metabolites and the observations of various assays are as follows,
Total RONS moieties. . . .decreased
Superoxide Dismutase. . Increased
Catalase. . . . . . . . . . . . . .Increased
Peroxidase. . . . . . . . . . . . Increased
How to correlate these results? If the total RONS moieties are decreasing then how there is increasing in the antioxidant enzymes activities? It would be the better if I get appropriate review paper in this concern.
Thank you for valuable explanations to all. One thing I have to add in my question is as follows. I have also did the Ferric Reducing Antioxidant Potential (FRAP), Total Antioxidant (TA), Superoxide Dismutase- like activity, phenolic contents and Nitric Oxide scavenging assays. In all these assay the exposed secondary metabolites have found to be acting as a good antioxidants.
So, how would I interpret these data with the in vitro experiments which I have mentioned previously?
I have performed a bioassay under laboratory conditions, where 2 benthic species were exposed to diesel oil-contaminated sediments. I only spiked the sediment with diesel oil at the beginning of the experiment, and thereafter I measured the activity of antioxidant enzymes at three different times. My controls showed some variation over time that was not significant for all but one enzyme. Since the animals have some stress because they are in laboratory conditions I'm wondering if normalising my enzyme response in the exposed treatments with regards to their controls is the way to go. What are the cons of this?
Glutathione is reduced mainly by glutathione reductase (GR). But in my catfish species we are not been able to identify/detect any gene sequence for GR. Although we have been able to sequence other glutathione dependent enzymes (i.e. glutathione S-transferase, glutathione peroxidase). So I am wondering whether there are some other enzyme or an alternative pathway of glutathione reduction present in these catfish species! Please help.
I have observed, at a certain dose nanoparticles promote seed germination. Some antioxidant enzymes get influenced, also pathogen attack on seed gets impacted. But what could be the exact mechanism behind the enhanced germination and/or vigour of seeds? Please suggest some clues.
Can anyone help me for antioxidant enzyme,and anybody give a standard protocol of CAT,SOD,GPx for fish tissues.
We produced the crude enzyme solution from white-rot fungi grown on apple waste. We did not do any purification and therefore the compounds in apple wastes are present in the solution. The target enzymes are laccase and lignin peroxidase. according to standard methods for laccase, we performed the oxidation of ABTS. But we saw a very small activity. Now I am thinking about the effects of antioxidants and polyphenolic compounds that are present in solution. Does anybody have any idea?
During oxidative stress,antioxidant defence system in erythrocytes is compromised. So, it could be suitable measure these enzyme activity in red blood cell. A lot of assay for SOD,CAT and GPx are reported.
I am estimating activity of CAT using spectrophotometer. The reaction mixture (1 ml) contains 850 uL of 50 mM phosphate buffer (pH 7.2), 100 uL of 200 mM H2O2 and 50 uL of enzyme extract. The absorbance is read at 240 nm at an interval of 5 seconds up to 120 seconds. Instead of linear decrease in OD, I am getting uneven pattern of absorbance. The OD decreases for 10-15 seconds, then increases and later follows zigzag pattern.
Upon doubt, I run a blank by adding only phosphate buffer and H2O2. I was expecting that I would get constant values or might observe slight decrease in absorbance. However, this time too, I observed zigzag pattern of OD.
I don’t understand why I am getting such pattern even when I am not adding the enzyme extract?
Solutions I tried:
(1) Use of quartz cuvette, not glass cuvette
(2) I replaced 200 mM H2O2 (prepared from commercially available 30% H2O2) with 3% H2O2 (sigma), but again same result.
(3) I don’t have doubt on sensitivity of spectrophotometer as it give correct readings during other assays
Hello I need to staining antioxidant enzymes in native page and I would like to have a protocol for each enzyme (CAT, POD, SOD, APX, GR). Could please someone share protocols with me? THANKS!
I am studying the response of Moringa seedlings to salt stress ,so I need to investigate the profiles of Proline, MDA and antioxidant enzyme : POX, CAT, SOD, and Ascorbate peroxidise. Can anyone tell me step-by-step procedures do that?
I am currently working on SOD enzyme assay in plasma. The protocol I used for SOD is incubating sample, PMS, NBT and NADH for 90secs at 30⁰C and adding acetic acid , butanol and measured absorbance at 560 nm. I am getting low blank value than test. I would like to know whether the absorbance of the blank without sample should be high or low. Could anyone please help?
Normally authors describes there role in ROS scavenging and believed that plant cells exposed to stress increase there activities in cell.
I have to perform antioxidant enzyme activity assay of different cell lines. I always seed Hep G2 cell line 30,000cell/ml. and after drug treatment I get only 80 to 100 microgram of protein only. so when assay were performed we get only catalase activity, no SOD and Glutathione peroxidase activity, so i want to know what should be total protein concentration needed for antioxidant enzyme assay...
I would need a protocol for measuring or identifying the availability of above mentioned antioxidant enzymes such as catalase, superoxide dismutase glutathione peroxidase using reagents that can be later determined using a spectrophotometer.
Would like to measure oxidative stress in the brain tissue. I am thinking to measure lipid peroxidation using MDA levels. If anyone has any suggestions, to measure oxidative stress in the brain tissue lysate using other parameters, please let me know.
- No intention to use commercial kit, as they are very costly and I am looking for in-house method.
- Paper link/PDF doesn't help as they briefly describe the method and does not provide full detail and I am looking for protocol for all details.
Hello! Maybe somebody knows how many cells (HEP G2 or THP 1) are needed to determine antioxidant enzyme activity (CAT, SOD, GR, GPx) and oxidative stress markers (like MDA, 8-OHdG, oxidized proteins,...)? Does anybody have experience using cultured cells?
Thank you for your help. Kindest regards