[bmilla35]

Hey guys and gals,

I'm currently finishing up a lab where we are purifying the acid phosphatase enzyme from wheat germ. I've calculated the total enzyme activity (units) for 6 fractions and then the "pure".

The manual clearly shows how to find all the previous calculations..and I just don't know how to do this. The unit is (Units/mg) of protein, so do I divide my Units by total protein? If that's the case for fraction one I would get a number of .018.

 If I haven't been clear I could try to upload the chart from Excel, just let me know.

[Yggdrasil]

Yes, the specific activity will be the units of activity divided by the total mass of protein in the sample.  After purification, the specific activity should increase because you are removing contaminating proteins that do not display enzymatic activity.

[bmilla35]

Great. Thanks for the confirmation!

[Babcock_Hall]

Beginning biochemistry students sometimes make errors in their calculations of total and specific activity because of problems with dilution calculations or other reasons.  Therefore, I suggest doing as many supporting calculations as possible.  For example, the total number of units before and after a chromatography step should either be roughly the same or should decrease slightly if the enzyme is easily degraded.

[Yggdrasil]

I'd say most columns should yield ~10-90% recoveries depending on the stringency of the purification.  If you recover significantly less than 10% of your activity after a column, you've either made a mistake in your calculations or in how you ran the column.

[bmilla35]

How can we tell if our "pure" fraction is 100% of the desired enzyme? Our procedure included fractionation (with salt and methanol with heat treatment) and then dialysis followed by an activity assay. The S.A. indicates it's much more pure than at fraction one, as the value rose from .14 to 3.18.

You would think there would be some some sort of salt residue, at least. Furthermore, what could be done to prove it? I was thinking another run through the fractionation and dialysis bag to see if the numbers change, but not sure.

As always help is greatly appreciated!

[Babcock_Hall]

Sometimes the specific activity can be compared with a literature value to assess purity.  SDS PAGE can also be used to show whether or not there are polypeptides of different molecular weight.  The presence of salt would only affect the specific activity if the enzyme were sensitive to changes in ionic strength (I am not certain that I understand your question).

[fledarmus]

Too many things are involved in specific activity for it to be a real test of purity of an enzyme. The best it can really do is tell you whether your preparation is consistent with somebody else's preparation, or whether it is consistent with the preparation you made last week. Other methods (like Babcock_Hall's SDS PAGE suggestion, other types of chromatography/detection combinations, or mass spec analyses) are used to determine purity.

[Babcock_Hall]

Fledermaus raises some good points.  Comparing specific activity with a literature value has some caveats that are worth understanding.  Not the least of these is that the literature may have a calculational error, a problem that beset a fellow graduate student.  One also has to duplicate the conditions of the assay for the comparison to be meaningful.