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?
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.
A reducing agent allows disruption of disulfide bonds and also avoid a-psecific aggregation related to disulfide bonds formation that could occur under oxidation during the breakage of cells. Very often it is good to add those agents in homogenizing buffer therefore to avoid this aggregation. Also DTT protects against some proteolytic attack. You can add either DTT , beta mercaptoethanol or cysteine to avoid these problems, it will not affect enzyme assay unless you are dealing with photosynthetic enzyme that are often redox regulated.
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.
Dear Adams,
As far as cysteine is concerned, I understand that it can be oxidized easily (by forming -S-S- links with another cysteine of polypeptide or free cysteine, dithiothreitol or mercaptoethanol), but I am not aware of any example or report where S of methionine has been shown to get oxidized (excuse me for my ignorance). As far as cysteine is concerned, it is simple to understand as two cysteine can be oxidized to for one cystine (i.e. 2 -SH give rise to one -S-S-), but I am unable to comprehend the same with methionine. I shall be highly obliged, if you can kindly send me any reference or report showing role of methionine of polypeptide/protein in redox reactions.
Here is a Wikipedia entry on methionine oxidation.
https://en.wikipedia.org/wiki/Methionine_sulfoxide
Thiol based reducing agent is not a great idea if your protein needs divalent metals for activity. TCEP [(tris(2-carboxyethyl)phosphine)] is a better substitute.
Overall, both disufides and metal determine the inclusion and choice of reducing agent.
Dear Adams
Thank you. It seems to be certain that methionine (in general) unlike cysteine will (i) not bring about cross link polypeptides/proteins; and (ii) not interact with cysteine/dithiothreitol/mercaptoethanol (i.e. it will not form -S-S- links).
We have been using cysteine, dithiothreitol as well as mercaptoethanol. But, I prefer cysteine and dithiothreitol over mercaptoethanol (due to peculiar odour of mercaptoethanol). We use one of them in extraction buffer to prevent aggregation of proteins due to disulphide linkages (when -SH of cysteine is free) during homogenization of samples (as pointed by Brigitte Gontero). During homogenization of samples we expect release of proteins/enzymes from all compartments of cells (and majority of them would be in hydrophilic environment.. However, if you are interested in extracting proteins from some specific compartments/parts such as cytosol or periplasm or membranes then you can take the suggestions of Adams or Kou.
In case of glutathion reductase assay, GSSG is used in the reaction mixture. So, what is the fate of GSSG in the reducing environment? Is there any negative effect of reducing agent on GSSG?
Dithiothreitol will reduce GSSG to GSH. Here is a reference:
http://www.jbc.org/content/248/2/707.full.pdf
If you have a cysteine in the active site, you should use DTT to keep it in the reduced form. Examples are cysteine proteases like caspases. If the cysteine in the active site becomes oxidized, the enzyme is no longer active
Hi Debaprasad
I have the same concerns as I used DDT in the homogenisation and separated the cytosolic fraction. I want now to assess the activity of ADH and GST in the cytosol. Did you have any obstacles in that?
Brigitte Gontero wouldn't the DTT affect the enzymatic assay by reducing the substrate ? May be I'm wrong and I'm not taking into account other factors.
It will depend first on how much you put in the homogeneization and how much the final concentration will be. It is always good to have reducing agent as they first chelate heavy metals that coudl be present in our buffer; 2) it will prevent the formation of disulfide bridge that could be deleterious for your enzyme as mentioned above. DTT is a powerful reducing agent so you don t need to put too much 1 to 2 mM will be enough. When you prepare it be careful about pH and freeze it (aliquot) immediately after you have done it. My recommendation in your case will be to do 2 samples, 1 with DTT and 1 without then you can be sure that you are in the best conditions as different enzymes behave differently.
dear Brigitte Gontero thank you very much for the complete answer. I will prepare two solutions and one of them I will take out the DTT with dialysis and check if there are any differences.
As a rule I try to avoid B-ME, as it can modify cysteines. An excellent result if you want to permanently avoid disulfide formation, but a disaster if your enzyme contains a critical cysteine.
Often this kind of modification is caught using mass spec. (PMC2911956) or X-ray crystallography (see map and model in structure of PMC3457536).
It actually totally depends on your protein. Many enzymatic assays require 1-2mM reducing agent (usually DTT) for the activity, while other enzymes need to contain disulfide bridges that could be reduced by DTT. You will need to try it, one sample with and one without DTT. Good luck
Thank you Josefa M. Alamillo I will give it a shot and see what happend!
The reducing agent like DTT may be added in the extraction buffer of enzymes. But it does not need to be added in enzyme assays except in case of nitrate redux tase wherein NADH is added in the assays as a reducing agent.
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