Re: [DIYbio] a quick question... Enzyme activity

I meant to post this too, it covers a few protein extraction/isolation
techniques except ELISA.
http://www.fgsc.net/neurosporaprotocols/How%20to%20isolate%20proteins%20final.pdf

This is specifically explainingsome chromatographic protein separation methods:
http://www.biotech.kth.se/courses/gru/courselist/BB2040_ENG/ChromMethods.pdf

On Mon, Sep 23, 2013 at 9:41 AM, Nathan McCorkle <nmz787@gmail.com> wrote:
> It sounds likely that the "per mg protein" is relating CAT[alase]
> activity to total protein, probably obtained through running a
> Bradford, Lowry or Biruet assay on a crude protein extract. This gives
> an approximation of protein content in solution, but is largely only
> reactive to the aromatic residues, so the effect depends not only on
> the protein concentration but also on the protein's amino acid
> content.
>
> If the "per mg protein" numbers were obtained via ELISA, or something
> like HIS-tag purification, then the "protein" would be mostly/highly
> pure CAT[alase].
>
> Seems that fresh weight is not preferred due to things like water
> content variation. If you know the fresh weight activity, and the
> activity of extracted protein, along with the activity of
> highly/mostly pure CAT[alase] then you would know what the
> concentration of CAT[alase] in fresh tissue, and you'd know the ratio
> of activity of the enzymes. The latter measurement would best be made
> with highly pure protein, but that's harder to do than crude protein
> extraction, and knowing the activity of CAT[alase] to total protein
> might often be enough to get your answer (say for example, is my GMO
> E.coli producing more insulin-production enzyme than pre-GMO). You
> don't need highly pure protein to answer that question because E.coli
> to E.coli will have similar total protein content, given they're the
> same or similar strain. So if you assume the pure enzyme's activity is
> constant, you can compare the activity amongst samples, if you don't
> know if the activity is the same (maybe one CAT[alase] gene is mutated
> and works slower than the other) you need to do a crude protein
> extraction and compare the activities... but ideally you'd purify the
> enzyme for each sample and compare highly-pure sample activities to
> determine if their rate was different or not.
>
> "
> Catalase activity was expressed per mg of extractable protein
> (specific activity) rather than on a per seed basis since the amount
> of extractable protein did not vary among the various seed samples (it
> was always close to 1.3 mg seed–1) whereas dry and fresh seed weights
> markedly changed during seed development. Indeed, sunflower seeds
> contain c. 15–20% albumins, which are extractable with the buffer used
> in this study, and c. 80% reserve protein (globulins, prolamins, and
> glutelins) (Lusas, 1985), which accumulate during seed‐filling, but
> were not extracted by the unsalted potassium phosphate buffer
> "
> http://jxb.oxfordjournals.org/content/55/396/475.full
>
> Here's a highly-cited paper on determining CAT activity:
> Catalase in vitro
> Hugo Aebi, Methods in Enzymology
> Volume 105, 1984, Pages 121–126
> http://libgen.org/scimag5/10.1016/S0076-6879%252884%252905016-3.pdf
>
> On Sun, Sep 22, 2013 at 12:07 PM, dragoljub dimitrijevic
> <buzda2@gmail.com> wrote:
>> Hi Brian,
>>
>> here is en example:
>>
>> I'm measuring CAT activity of Pisum sativum leaf samples that were treated
>> with different UV. Samples vary by protein content and by measured enzyme
>> activity (Abs. H2O2 @ 240nm). Measured enzyme activity gets me to activity
>> per ml (U/ml) which can be expressed per gram FW (U/g FW) or per mg protein
>> (U/ mg protein)  (fresh weight and protein content per ml are already
>> measured). If measured CAT activity is expressed per gram of FW I get one
>> set of data among exp.groups and if expressed per mg of protein then it's
>> completely different set of data leading to different conclusion. Sometimes
>> in papers enzyme activity is expressed per gram FW and sometimes per mg
>> protein. What are (dis)advantages of each method? I searched for difference
>> and I could not find it...
>>
>>
>>
>>  Thanks!!!
>>
>> Dragoljub
>>
>>
>>
>>
>>
>>
>> On Sun, Sep 22, 2013 at 2:11 PM, Brian Degger <brian.degger@gmail.com>
>> wrote:
>>>
>>> can you give an example?
>>> its likely that the fresh weight is not fully purified mg of protein.
>>> this figure might explain it
>>> http://www.ncbi.nlm.nih.gov/books/NBK22410/table/A465/?report=objectonly
>>>
>>>
>>> On Sat, Sep 21, 2013 at 3:42 AM, dragoljub dimitrijevic <buzda2@gmail.com>
>>> wrote:
>>>>
>>>> Hello world :-)
>>>>
>>>> I would appreciate a lot if somebody can tell me what is the trade-off
>>>> when enzyme (CAT) activity is expressed per gram fresh weight Vs. per mg
>>>> protein ?
>>>>
>>>> I get very different results and don't know how to interpret difference?
>>>>
>>>> Thanks in advance :-)
>>>>
>>>> Dragoljub
>>>>
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>>>
>>>
>>>
>>>
>>> --
>>> ----------------------------------------
>>> Brian Degger
>>> twitter: @drbrian
>>>
>>> http://makerspace.org.uk
>>> http://transitlab.org
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>
>
>
> --
> -Nathan



--
-Nathan

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