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Calculating specific enzyme activity

specific enzyme activity

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#1 Mwanafunzi

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Posted 04 April 2015 - 06:52 AM

Hi!

I am writing my bachelors thesis and I am a bit stuck and feel unsure..unsure.png . I am looking at activity of few metabolic enzymes in fish larvae (Citrate Synthase and Pyruvate Kinase). Since my extracts are not pure I have to calculate specific enzyme activity. The enzyme activities are in U/g wet mass (U = µmol min-) and protein content is in µg/mg. So I have to divide enzyme activity by protein content..right? but I am not sure of the final units.

Here is an example for one sample for the enzyme Citrate synthase:

Enzyme activity = 6.728 U/g

Protein content = 24.222 µg/mg

Do I have to do a conversion of one of the factors to have uniform units?..how?

 

Is someone kind enough to  sort out the units for the above as an example?...any useful sources where I can read on such a topic?

 

I will really appreciate your contributions

 

Cheers!

 

 

 


Edited by Mwanafunzi, 04 April 2015 - 06:58 AM.


#2 mdfenko

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Posted 06 April 2015 - 03:06 AM

did you mean ug/ml for protein content?

 

specific activity is units/mass of protein, it is often used to determine purity of the enzyme in question (when the pure enzyme specific activity is known).

 

what you are calling "enzyme activity" appears to be specific activity.


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#3 Mwanafunzi

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Posted 06 April 2015 - 03:33 AM

Thank you for your reply mdfenko. The units were µg/ml for protein but after multiplication with respective dilution factors, it changed to µg/mg (where mg is the weight of the initial sample used).

For the 'enzyme activity', U/g (where g is the weight of the sample used and not protein content). Isn't specific activity supposed to be derived from enzyme activity (U/g) and protein content?...I haven't done that yet, so I think, I still don't have specific activity yet. Or may be I am a bit confused ..unsure.png

In my case I am not determing the purity of the enzyme, but rather using enzyme activities as indicators of possible tolerance to environmental condtions like hypoxic waters (waters with low oxygen levels). So may be I don't need to calculate specific activity after all?



#4 mdfenko

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Posted 06 April 2015 - 03:43 AM

enzyme activity is units. specific activity is units/mass of protein.

 

when you say ug/mg are you saying you have ug of protein per mg of tissue?

 

if that's the case then you have to convert grams of tissue to mass of protein (g x (ug/mg) x 1000) (the 1000 is to convert grams to mg).

 

this wikipedia page may give you more insight: enzyme assay.


Edited by mdfenko, 06 April 2015 - 03:47 AM.

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#5 Mwanafunzi

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Posted 06 April 2015 - 04:33 AM

You got my problem right! There is this calculation sheet that we are using in the lab and it wasnt so clear to me in some parts. Like we had to calculate enzyme activity in Units per gram of tissue...yet we weigh our tissue samples in mg (so we have to convert mg to g). So if I had stuck with mg...and get the enzyme activities in units per mg of tissue then I wouldn't have to start converting back to units per mg again like you put it there. Thank you for help and the link too.

But just to be sure, like in ur latest explanation.

In the example I gave above above:

 

Enzyme activity of sample x = 6.728 units/g (units per g of tissue) [(the tissue weight is 0.00635g...initially weighed in mg (6.35mg))]

Protein content of sample x= 24.222 µg/mg (µg of protein per mg of tissue) (tissue weight: 6.35mg).

 

Whats the easiest way to solve that one and calculate specific activity?...Can you help in this one as an example?

 

Thank you for your help so far



#6 mdfenko

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Posted 06 April 2015 - 04:52 AM

okay...

 

you have 6.728 units/g tissue (which is a sort of specific activity itself) and used 6.35 mg of tissue in the assay to determine this. to match units, 6.728 mU/mg tissue x 6.35 mg= 42.7228 mU from the assay.

 

you have 24.222 ug protein/mg tissue. 6.35 x 24.222= 153.8097 ug protein in the assay (~0.154 mg).

 

42.723 mU/0.154 mg protein= 277.422 mU/mg protein=~ 0.277 U/mg protein


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#7 Mwanafunzi

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Posted 06 April 2015 - 05:26 AM

Perfect and now I understand the concept...and thank you for taking your time to help.

One last question: in the explanation you gave where you have ''to match units, 6.728 mU/mg tissue x 6.35 mg= 42.7228 mU from the assay''

why have 6.728mU/mg tissue and not 6.728mU/g tissue? (i.e why mU per mg tissue and not mU per gram tissue...?..the activity is in per gram tissue)...sounds dumb on my part..but kindly explain anyway.



#8 mdfenko

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Posted 06 April 2015 - 05:36 AM

mU/mg=U/g

 

you were working with mg amounts so i simplified the units for your purpose.


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#9 Mwanafunzi

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Posted 06 April 2015 - 06:33 AM

Problem solved...thank you very much. And from what you showed me there in mU/mg=U/g I am already playing around:smile.png

 

Enzyme activity of x= 6.728U/g =6.728 mU/mg

Protein content of x= 24.222 µg/mg

 

Specific activity  = (6.728 mU/mg)/(24.222 µg/mg)

                         

                         =  6.728 mU/mg x mg/24.222 µg  (mg cancel out)

 

                         =  0.27776 mU/µg

Close to what you calculated above...only if mU/µg = U/g...thinking.

 

 

Thank you sir



#10 mdfenko

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Posted 06 April 2015 - 06:42 AM

mU/ug=U/mg


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#11 Mwanafunzi

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Posted 06 April 2015 - 07:04 AM

Thank you, and by 'mU' the 'm' stands for 'micro' or is it 'milli'? I promise this should close my 'bothering' you. smile.png



#12 mdfenko

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Posted 06 April 2015 - 07:19 AM

"m" means milli

 

"u" or "µ" (micron) means micro


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#13 Mwanafunzi

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Posted 06 April 2015 - 08:01 AM

My problem is sorted out well now. Thank you very much sir.smile.png






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