I am studying Bovine alkaline phosphatase stability under different conditions. I checked the tertiary structure stability using Intrinsic fluorescence (Tryptophan) and could see that it had lost its tertiary structure. However, when I ran an activity assay using pNPP, the activity seemed unchanged and almost equal to standard ALP solution. So is it possible for a protein to lose its structure but retain its activity? Thank you in advance.
Dear Shashwati,
To my knowledge, the destruction of the tertiary protein leads to lose of its activity;
The tertiary structure can be altered by a number of factors that will interfere with the molecular processes that hold the structure together. For tertiary structure, these includes changes in temperature, pH, and ionic strength. Since the function of a protein is dependent on its structure and any factor that affects the structure will also affect the activity of the protein. Thus heating a protein can affect its structure and thus its activity.
The disruption of the protein structure (and thus its activity) by some "unnatural" condition (heat, pH, etc.) is known as denaturing the protein. This denaturation is irreversible such as in the case of boiling eggs.
If in your case Bovine alkaline phosphatase still active it means one of both: (1) you have not fully destroyed the tertiary structure of the phosphatase or (2) the fluorescence experiment that you have carried out some how failed.
I suggest that you run the destruction experiments for 2-3 times to validate the unexpected finding you reported.
Hoping this will be helpful,
Rafik
Sound advice from Rafik. Are the buffers you are using to measure the Trp flouroscence and your assay the same? AP needs metal ions to adopt the catalytically competent structure. Is it possible the buffer for the Trp assay was devoid of metal ions or had a chelating agent?
Shashwati,
A partial unfolding of an enzyme is possible without the total loss of biological activity - it all depends on the specific enzyme and the method used to partially denature it.
Looking simply at change in the intrinsic Absorption of Tryptophan is not a sufficient evidence that your enzyme (which is a dimer, if memory serves me correctly) is fully denatured. To accomplish denaturation, you will need something more drastic than what you may be using, such as 6M Guanidine HCl or 8M Urea.
Alternatively, you could simply suck the Zn++ out and the biological activity will virtually disappear even if the conformation of the rest of the molecule remains largely intact. ;)
Some proteins don't loose their activity while loosing their secondary and tertiary structures. For example, lysozyme exhibits unfolding while adsorbing, but has
been shown to maintain enzymatic activity upon attachment to solid surfaces.
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