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Topic: In our lab I see no trend in total enzyme activity- is this right?  (Read 3970 times)

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Offline Kalibasa

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We purified wheat germ to study acid phosphatase and assayed it at each step for enzyme activity and protein concentration. In our data I am seeing a definite decrease in the total amount of protein (which makes sense, since we are progressively filtering out unwanted proteins) and a definite increase in specific activity (which also makes sense- the enzyme is becoming more pure). But we are also asked whether or not we see a trend in total enzyme activity, and I simply don't see one. 

I checked the literature and found accepted data that is similar, so I'm guessing this must be right. But I don't understand, conceptually, why there would be no trend. Why would total enzyme activity also not be increasing?

Thanks!

Offline Yggdrasil

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Re: In our lab I see no trend in total enzyme activity- is this right?
« Reply #1 on: March 04, 2010, 12:26:31 PM »
Although you are purifying the enzyme, each step of the purification is not perfect.  At each step you will lose some enzyme, but the hope is that you will lose many more of the contaminants.  Certainly during a purification, you should not see an increase in the total amount of enzyme activity, because that would suggest that enzyme is appearing out of nowhere (or, perhaps more realistically, you are purifying the enzyme away from some inhibitor).

In general, when designing an enzyme purification strategy, you need to balance yield (how much enzyme you get out in the end) versus purity (how few contaminants there are).  In general, adding more purification steps will decrease the yield but increase the purity.

Offline Kalibasa

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Re: In our lab I see no trend in total enzyme activity- is this right?
« Reply #2 on: March 04, 2010, 11:39:23 PM »
It makes sense that you shouldn't be seeing an increase in the total amount of enzyme activity, and yet these were the numbers from nearly the same experiment in a well-respected book:

171
160
72
121
86
133

Our lab manual specifically wanted us to compare to these values, and the protein concentrations and specific activities were perfect, so I'm guessing this must be right. But it just makes no sense to me. Even with the purification steps being different, how can this possibly be right?

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