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biochemical assay: induction


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4 replies to this topic

#1 kentz19

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Posted 15 April 2009 - 02:59 AM

Hi,

Is it usual to get increase in enzyme activity during in vitro biochemical assay? What I understand so far, in vitro biochemical assay is meant for observing the inhibition of enzyme activity by test compound. In my case, I get increase in enzyme activity at lower concentrations every time I conduct my experiment. I'm using herbal extract and absorbance to detect formation of product. Can I explain the increase of enzyme activity is due to increase in turn over rate of enzymes by the herbs at lower concentration? I didnt find any article discuss about such thing.

TQ

#2 mdfenko

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Posted 15 April 2009 - 06:17 AM

there are a few possible reasons for increased activity at low concentrations of your herbal treatment.

you could be experiencing pH change, addition of some cofactor or substrate, change of salt concentration, presence of some other factor that stimulates activity at low concentrations, your assay may not be optimized.

some inhibitors have been shown to stimulate at very low concentrations.

are you seeing inhibition at higher concentrations?
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#3 kentz19

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Posted 17 April 2009 - 02:35 AM

there are a few possible reasons for increased activity at low concentrations of your herbal treatment.

you could be experiencing pH change, addition of some cofactor or substrate, change of salt concentration, presence of some other factor that stimulates activity at low concentrations, your assay may not be optimized.

some inhibitors have been shown to stimulate at very low concentrations.

are you seeing inhibition at higher concentrations?

Dear mdfenko,

My extract show significant inhibition only at 500 ug/ml (71 % GST activity). Further observation with higher cocentration is not possible due to deviation from Beer Lambert law. GST activity is 123% (0.01 ug/ml) and 114 % (10 ug/ml). For other concentration, only slight reduction in GST activity. For GST activity that exceed 100 %, can I assume the inhibition equal to 0 as for 0.01 and 10 ug/ml ? For 500 ug/ml, the inhibition is only 30 %. Can I find an IC 50 if the inhibition value not exceed 50%? How about if the extract dont show any significant inhibition (the highest inhibition is only 9 %), can I find the IC50?

TQ

#4 mdfenko

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Posted 17 April 2009 - 09:40 AM

dear tq,

ic50 is the concentration of inhibitor at half the maximum inhibition so, yes you can determine ic50 even if the inhibition tops out at 9%. in this situation 9% is maximum inhibition.

see the wikipedia page on ic50
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#5 DRT

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Posted 20 April 2009 - 11:05 PM

dear tq,

ic50 is the concentration of inhibitor at half the maximum inhibition so, yes you can determine ic50 even if the inhibition tops out at 9%. in this situation 9% is maximum inhibition.

see the wikipedia page on ic50


I think this interpretation of IC50 should only be used when there is clear evidence that the activity has maxed out e.g. a good sigmoidal curve up to 9% inhibition. In this instance it looks like there is just minimal inhibition and I would use something like IC50 > 500ug/mL (or maybe IC10 > 500ug/mL).




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