It might be due to the pI of the protein itself, you can load the protein sequence into a web page like http://web.expasy.org/protparam/ which will calculate the isoelectric point for your recombinant protein: the isoelectric point is the pH at which your protein will be neutrally charged and likely to be more stable. In addition you have to consider that in the cytoplasm there are salts which will neutralise the protein charge, while in solution to preserve stability you need to change the pH. I hope this clarify it for you.  

Hi Mervi, this is more of an evolution question than a structure/function question.

Natural selection has been constantly working to favor beneficial mutations that improve an organisms ability to survive without drastic changes to its physiology and energy consumption.

Metabolic proteinases like trypsin, chymotrypsin and elastase have a pH optimum activity above pH 7.4. You might ask the same question: Why is that?

If you accept natural selection as the main driving force to improve survival of an organism, the answer is that these enzymes do their jobs at physiological pH. They might be better at doing their job at higher pH, but that would require a total change in the buffer system of the body that relies on bicarbonate to keep the pH constant throughout the body.

As for your cytosolic enzyme, its activity at physiological pH is clearly enough for it to do its function.

Domenico, I know the Expasy pI/Mw-calculation site, my enzyme actually shows acidic pI (~5.3). Yes, indeed in the recombinant protein assay we do not use salts that are normally present in the cytoplasm.

Do you/anyone know what is the best buffer composition to mimic cytosolic salt & pH conditions?

That will be PBS, Phosphate Buffer Saline.

Thank you all for your answers.

Domenico, PBS does not contain Mg or Ca, which are to my knowledge present in the cytoplasm. And how about glucose?

you can find PBS with Calcium magnesium and Glucose:

https://www.thermofisher.com/order/catalog/product/14287072

there are also plenty of recipes around to make it yourself!

Domenico, I usually prepare buffers by myself (inlcuding PBS with or without Ca/Mg), it is much cheaper that way.. and I know many buffer compositions.. 

but what is the buffer that most closely mimics the conditions of cytosol?

PBS with Ca,Mg and glucose?

It all depends on what you want to do with it, I'll suggest you try first with simple PBS, then you can check some literature and maybe explore different recipes, but, to my knowledge, cytosol is also viscous, so to replicate its behaviour you need PEG as well, you might want to read something about Cell Free expression systems to have an idea. Anyway without further information is difficult to help.

Calcium is at very low concentration in the cytoplasm. It is sequestered in organelles. Mg is present in several mM concentration in the cytoplasm.

The cytoplasm is a reducing environment, with several mM glutathione. You should therefore put a reducing agent in your buffer (2-mercaptoethanol, dithiothreitol, or glutathione).

Hi there,

To answer the question I presume the enzyme activity in the cellular context is enough to ensure its function in the cell (btw how different are the enzyme activities at the 2 different pHs?). I think the right question is what would be the cell fitness if cytosol was actually buffered at pH 9 (ie. optimal for this particular enzyme)?

I experienced something similar. My protein had pH optimum somewhere around 11 (although I think I haven't measured at higher pH). Unfortunately the project was terminated (also because of this fact), so I did not elaborate further.

Although I had enzyme from native source, partially purified; not recombinant.