I am working on enzyme production and want to study its kinetics. To stop an enzymatic reaction I need to add KCN. Due to potential toxicity, I don't want to use KCN, so please suggest some other enzyme inhibitors that have little or no toxicity..
Depending on the type of enzyme, one uses an inhibitor. Please disclose what enzyme you are targeting then only an appropriate inhibitor for it can be suggested, For example if your enzyme is a metallo enzyme with Mg or Ca then one can go for EDTA or EGTA as chelating agent.
Or you can change the pH dramatically by either adding a concentrated acid or base.
You can also increase the temperature, or add EtOH to denature the enzyme. It all depends on the enzyme that you use
Thanks for your suggestions...will try them...
My enzymes belongs to the group of oxidases which does not require metal atom or organic co-factor for catalysis..
When KCN is used to inhibit oxygen uptake by membrane-associated oxidoreductases that are coupled to a respiratory chain, 0.05-0.1 % Triton X-100 can sometimes provide an effective, non-toxic alternative.
You can use sodium azide as its a known inhibitor for cytochrome oxidase instead of KCN. Hope it serves your purpose.... Best regards
Deepti
It is rare that your oxidases have no metal and they are inhibited by KCN. KCN is an inhibitor of aerobic oxidation and there are cytochromes with Fe and Cu involved. Anyway, if your problem is the toxicity of KCN, try other metal chelators not so toxic, sucha as nitrilotriacetic acid. Azide is also effective, but toxic. Probably EDTA is not appropriate, but you can also try.
Nonionic detergents may be employed, as Dr Pronk suggested. Tween 80, saponin, cholic acid have also been utilized. Other potential enzyme inhibitors could be 3-aminotriazole, bathocuproine salts, potassium ethyl xanthate, ortho-phenanthroline, etc. A plethora of enzyme inhibitors exists (examples: those that target serine proteins; sulfhydryl reagents; disulfide reducing agents). As an aside, mitochondrial monoamine oxidases (MAO; flavin containing) from certain tissues contain noncatalytic iron, and incubation with ortho-phenanthroline has no discernible effects on enzymic activity. Conventional polarographic assays for determining MAO in mitochondrial fractions use 100mM KCN. On the other hand, some MAO preparations are susceptible to KCN (>mM). Metals do not appear to be essential for activity in certain urate oxidase preparations; nevertheless some urate oxidases may be inhibited by KCN. Non-oxidases, for example, lysophospholipases display no obligatory requirement for any cofactors, and yet there are instances wherein KCN strongly inhibits certain lysophospholipases.
there are several parameters that effect enzyme activity: pH, temperature etc.
-adding acid or base
-decreasing the temperature
- increasing salt concentration
- using some drugs i.e. oxidase inhibitors (easily obtained from pharmacy)
or using the combination of all these..
Many many thanks for the valuable suggestions...will surely try these...
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