We recently have found that nanoparticles can inhibit enzymes. But we don't know the exact mechanism and whether it is an irreversible inhibition or not.
I would measure the activity of a given concentration of enzymes, and then titrate in gold nanoparticles until the activity is again constant, or perhaps zero. That will give you the molar ratio of enzyme to gold nanoparticles under these conditions. The concentration of the gold nanoparticles can be monitored very well by UV-vis spectroscopy unless the particles aggregate, which would initially be manifested by a colour change from red to blue. I suppose this does not happen in your experiment. It is then not normally possible to free the enzymes again, unless they survive the conditions under which you can dissolve the gold nanoparticles. These are either dilute agua regia (likely to destroy the enzyme completely), iodine (may be ok) or cyanide (probably fine but don't kill yourself!). Alternatively, you could try ligand replacement by adding a large excess of, say, dithiothreitol. This may liberate the enzyme. In any case, it is not too clear to me what you will learn from having recovered the enzyme.
I would measure the activity of a given concentration of enzymes, and then titrate in gold nanoparticles until the activity is again constant, or perhaps zero. That will give you the molar ratio of enzyme to gold nanoparticles under these conditions. The concentration of the gold nanoparticles can be monitored very well by UV-vis spectroscopy unless the particles aggregate, which would initially be manifested by a colour change from red to blue. I suppose this does not happen in your experiment. It is then not normally possible to free the enzymes again, unless they survive the conditions under which you can dissolve the gold nanoparticles. These are either dilute agua regia (likely to destroy the enzyme completely), iodine (may be ok) or cyanide (probably fine but don't kill yourself!). Alternatively, you could try ligand replacement by adding a large excess of, say, dithiothreitol. This may liberate the enzyme. In any case, it is not too clear to me what you will learn from having recovered the enzyme.
Proteins bind to nanoparticles by charge intearaction and binding is reversible. Regarding the activity of enzymes upon binding , depndends what size of nanoparticles you are using e.g 20 to 30 NM gold particles are quite bigger than average size of proteins ! It is not necessary nanoparticles will inhibit enzymatic activity.
Depending on the length of any linker molecule used to immobilize your proteins on the surface, it may not interfere with the enzyme function. For instance, restriction enzymes still work within the confines of DNA-assembled nanoparticles. If you can get the protein far enough away from the NP surface, the enzyme should still be able to function.
Alternatively, you can try adding salt to the solution to minimize the influence of the nanoparticle, but too much salt will precipitate everything out of the solution.
It may also have to do with whether disulfide bridges in your enzyme are critical to its activity, and if those disulfides get cleaved when the proteins form Au-S bonds attaching to the AuNP surface.
you adsorb an enzyme on a metallic surface without linker, thus you change its conformation so it is not surprising that the enzyme doesn´t work anymore. You may also be interacting with thiols, this "kills" your protein... It is also possible that the nanoparticle causes electronic perturbation in the protein You have many sources of problems!
Related publication is attached.
Article Spectrophotometric evaluation of gold nanoparticles as red-o...
You may remove particles after contact with enzyme preparation, e.g. by centrifugation.
Then you should be able to analytically determine amount of enzyme in solution, e.g. by UV absorption or a more specific method. Then you can compare enzyme activity before and after contact with nanoparticles, but you have to ensure identical solution conditions, pH etc.
Binding of ions released by nanoparticles may cause problems. Therefore you should also determine content of these ions after adsorption.
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