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Topic: Enzyme Assay Question  (Read 7012 times)

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Offline Einherjar_PT

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Enzyme Assay Question
« on: May 23, 2008, 12:02:20 PM »
In a kinetic study of  yeast invertase (EC 3.2.1.26) protocol, the enzyme was first prepared in a Tris-HCL (ph=7,5) 0,01M Buffer, and then added to the substrate, dissolved in distilled water and prepared with a Sodium Acetate (ph=4,5) 0,2M Buffer.
Why was the enzyme solution prepared in a different buffer?

Offline Gerard

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Re: Enzyme Assay Question
« Reply #1 on: May 24, 2008, 01:55:12 PM »
In a kinetic study of  yeast invertase (EC 3.2.1.26) protocol, the enzyme was first prepared in a Tris-HCL (ph=7,5) 0,01M Buffer, and then added to the substrate, dissolved in distilled water and prepared with a Sodium Acetate (ph=4,5) 0,2M Buffer.
Why was the enzyme solution prepared in a different buffer?

-it can be trace to the properties of the enzymes, recall that enzymes are proteins and a slight changes in the pH will cause the enzyme to denature that is why a pH of 7.5 is needed as you can observe the substrate is prepared in an acidic buffer this is ,i think, to prevent partial hydorlysis of the substrates..

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Offline Einherjar_PT

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Re: Enzyme Assay Question
« Reply #2 on: May 24, 2008, 03:49:39 PM »
The optimal pH of the enzyme is 4,5, that's probably why the substrate uses a 4,5 buffer.
But yes, the use of the 7,5 buffer in the enzyme may be to avoid protein denaturation...
Thanks for the insight

Offline JGK

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Re: Enzyme Assay Question
« Reply #3 on: May 26, 2008, 07:40:52 AM »
the reason it was prepared in pH 7.4 buffer is that is the pH commonly regarded as  "physiological".  Reactions monitored at this pH will more accurately mimic "in-vivo" kinetics.
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Offline Yggdrasil

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Re: Enzyme Assay Question
« Reply #4 on: May 26, 2008, 10:06:21 AM »
Although pH 7.4 is usually the natural pH at which many enzymes work, there are many enzymes whose natural environment is a different pH.  For example, consider the proteases in the stomach (e.g. trypsin) which act naturally at very low pH.  Similarly, enzymes in the intestines naturally work at a slightly basic pH.  Subcellular compartments (e.g. lysosomes) also have pHs that differ from physiological pH and enzymes that have evolved to work at the pH of that organelle.

The fact that invertase works optimally at pH 4.5 makes me think that it may be a lysosomal protein, consistent with its role in degrading sugars.

Offline Gerard

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Re: Enzyme Assay Question
« Reply #5 on: May 27, 2008, 09:25:09 AM »
now that ypu are opening that up it makes me realize the opposite of trypsin which is pepsin...
and it works at the opposite pH of trypsin...
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