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I am trying to do coursework, on whether a chemical is a competitive or an non competitive inhibitor of amylase. Only problem is, i don't know what chemical i should use. Can any1 suggest a chemical that will be easily available in a school lab, i have tried googling it, but it keeps coming up with weight loss pills!! I was suggested Iron 3 Chloride, any good?
Amylase (at least, human salivary amylase) is a metalloenyzme which binds 1 Ca per molecule of enzyme. Iron(III) or Iron(II) salts might be inhibitors, so might Barium(II) or possibly Cadmium(II) salts—but watch out with the latter especially; it binds strongly to sulfhydryls and might simply inactivate the enzyme. I know amylase has a number of disulfide bridges, which probably won’t react with Cd, but if there is a crucial free cystine residue, well, you may get inhibition, but exactly what the kinetics will be, I wouldn’t care to predict. Maybe you would get what is called dead-end inhibition? I would try and keep the metal ion concentrations low-ish. If your amylase is being used at, let's say, 1-10 micromolar, then try and keep your metal concentration to 5-50 micromolar and less to try and minimize any tendency (if there is one) for the metals to precipitate the protein. You want to inhibit the enzyme, not drive it out of solution.
There are small oligosaccharide inhibitors of amylase, too. These you probably won’t find laying around a typical undergraduate laboratory. You might consider making some sort of aqueous extract of seeds, seedlings, or sprouts to see if you can get anything that way. You won’t be able to characterize them very well, but you may still be able to see that if you prepare the extracts in the same way, you get inhibition of anylase activity, or that different types of seeds, etc. prepared the same way give different degrees of inhibition. If you just happen to have a selection of carbohydrates laying around, you might try them, though I’m not sure you will get spectacular amounts of inhibition—maybe at very high concentrations? I don’t know how well those things may (or may not) work. Try things like ribitol or arabinitol (any kind of –itol you might have) or maybe even citric acid or soduium citrate. Tris buffer has also been reported to inhibit amylase, but I’m not sure which amylase that was. If you start using things like citrate or citric acid, you will also have to make sure that your solution is buffered adequately (typically 100 mM at pH whatever--I would stay away from Tris buffer, but imidazole maybe? or HEPES?
There are baby oligosaccharide inhibitors of amylase, too. These you apparently won’t acquisition laying about a archetypal undergraduate laboratory. You ability accede authoritative some array of aqueous abstract of seeds, seedlings, or sprouts to see if you can get annihilation that way. You won’t be able to characterize them actual well, but you may still be able to see that if you adapt the extracts in the aforementioned way, you get inhibition of anylase activity, or that altered types of seeds, etc. able the aforementioned way accord altered degrees of inhibition. If you just appear to accept a alternative of carbohydrates laying around, you ability try them, admitting I’m not abiding you will get amazing amounts of inhibition—maybe at actual top concentrations? I don’t apperceive how able-bodied those things may (or may not) work.
Last edited by canalon on Thu Jun 25, 2009 4:29 pm, edited 1 time in total.
Reason: Spamming in sig
taking alpha-amylase (eg: salivary amylase in human) you can easily get it by extraction from common beans (Phaseolus vulgaris).
But the purification techniques may be complex process, which include heat treatment in acidic medium and chormatography technique.
Student of M.Sc. Microbiology,
GITAM Institute of Science,
GITAM University, AP, India
e-mail: [email protected]
6 posts • Page 1 of 1
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