Science Buddies: "Ask an Expert"

I want to do a science project measuring how pH affects protein solubility or how it affects protein aggregation. However, I don't know how to test this at home. I would really like some links or suggestions of how to do this.

Thanks!

I've moved this to the Life Sciences forum, as I think you're more likely to get useful answers from the people there regarding the field of proteins.

Hello there,

That sounds like a very interesting and cool idea!! But I'm not exactly sure how you can do an experiment on that easily and at home...You would need the actual enzyme (protein) and have different pH level solutions to test them in. Every protein has its own optimum pH level and if it is outside that pH level then protein aggregation will happen and the enzyme will not function properly and will probably deform. Most proteins will probably have an optimum pH of around 7 which is neither basic nor acidic which is approximately what it is in human body.
One example is pepsin which is an enzyme used in digestion and will only activate at very low pH levels which are only found in the stomach....

I hope this helps!
Prasad

Thank you for your advice! I was wondering more on how to measure this. How can you measure the solubility or aggregation of proteins? Is there a device or procedure?

iperelman,

One easy way to test this would be to purchase a water soluble protein, such as egg albumin. If you use whole eggs, while they contain alot of albumin, they also contain fats, which are not water soluble. Using an egg albumin powder will allow you to weigh (or measure by volume, such as tablespoons) the ppowder out before stirring it into the solution. For each solution at different (known) pH, you can record how many grams (or tablespoons) of egg albumin powder you could stir in before it would no longer go into solution.

Hello there,

This sounds like an extremely interesting project. I did some searching online, and I found a video of a project that measured the effects of pH on the solubility of casein. You might want to take a look at it: http://jchemed.chem.wisc.edu/JCESoft/CC ... /MOVIE.HTM

There is also an interesting article on wikipedia on protein precipitation, and it describes the effects that pH has on the repulsion and attraction forces that act on proteins in solution: http://en.wikipedia.org/wiki/Protein_precipitation

If you would like an advanced paper on this topic, have a look at this: http://www.springerlink.com/content/v56 ... lltext.pdf

Hope this helps, and Good Luck,
Nithin T

This all helps me a lot!I'd just been about to post a question on different protein powders because I have changed my project a bit. I am now researching how different proteins dissolve in salt. The video about casein helped, because I instantly researched the protein and found a few others. These are some or all of the proteins that I am using: egg albumin, casein, whey and possibly soy protein. Are there others? As well, I need to purchase these ingredients and am having trouble finding them without any additives. They are often accompanied by salt, which is something that I don't think I can use in my experiment.

Hi!
Sounds like a great project! I'm not quite sure how you're going to measure how different proteins dissolve in salt, because protein powders will not truly dissolve unless you put them into some type of liquid solvent. If this is what you want to do, I would still suggest you go back to measuring how different proteins dissolve in water. Salt is very hard to use for your project because egg albumin naturally has salt in it, as does casein, so it will be nearly impossible to find these protein powders without salt in them already. Hope this helps, good luck!

I have tried to do an experiment using the procedure that sunmoonstars presented, and am experiencing a difficulty, I don't know exactly when it begins to aggregate. There were clumps at some point, but I was able to dissolve them by using a spoon, or simply shaking the container. I used whey protein, and tried to dissolve it in water(no salt, vinegar, soda etc.). When exactly am I supposed to note down that it aggregates? When it gets more dense in colour? When it forms clumps? I also tried adding a bit of vinegar with no results. Then I left it alone for several minutes, without shaking it and noticed that, there was a layer of lighter liquid on the top, and a denser, yellower liquid on the bottom? Is this a sign of aggregation?
Thanks!

Hello,

That's great you had the opportunity to start your experiment already. How much powder albumin did you have in the water? I found a web reference that says the albumin could be soluble as much as 1g/ml (1 gram albumin powder in 1 milliliter of water), but I can't be sure how reliable that figure is.

If you can still break the clumps up with a spoon and they go into solution, I would not consider that an aggregate. The protein will clump or aggregate when no more will go into solution - and it may be heavy enough to fall to the bottom of your water sample. This is actually what you are seeing in the glass when you let it stand untouched - the albumin is falling near the bottom of the glass and appears darker down there than up near the top. In that sample, you have probablly gotten very close or exceeded the solubility point of the albumin in water. How much albumin was in there (and how much water)?

We didn't use egg albumin, we used another protein powder called whey. We had 10 ml of water and at the point where we saw the heavier liquid at the bottom there was 10 ml of whey powder. We also tried putting the container in hot water for a long time, with no results. I'd read on the internet that temperature affects protein solubility. When you say:"The protein will clump or aggregate when no more will go into solution" what exactly do you mean? What gives us a clue that "no more will go into the solution"? Will I see something? Because from the beginning, (1 ml of whey powder in 10ml of water) the colour of the water had changed to murky yellow. I need to know how to determine when the whey powder is soluble, or is aggregating.

Hi again,

Yes, the solubility is based on temperature as well as pH (you can read more about that here - http://cascavel.ufsm.br/revista_ccne/oj ... le/186/193).

What I mean is that the powder won't solubilize, meaning it floats around in the water. You should be able to see it with your eyes. If you describe the water as 'murkey', then the whey is not all in solution.

Here is how I would set up the next trial. I found the paper I linked above that the solubility was ~11mg/ml. You should be weighing the whey powder on a scale in g or mg, so you can figure the solubility in g/ml (or mg/ml). I would weigh out 5 mg on some weigh paper. I would stir 5mg whey into 5ml water (this is a concentration is 1mg/ml). Stir it until the water is clear. It may have color to the water (like brownish or yellow), but you should be able to see right through it and no particles should be floating around - this is what completely solubilized protein looks like. Now add 5mg more to the same water and stir it (this is a 2mg/ml concentration). Again, it should go completely into solution. the color may get stronger as you add more protein, but it should keep going into solution with no particles floating around. Continue to do this, 5 mg at a time, until you notice you can't get the floating peices to dissolve, even if you stir it alot. Then you can see what it looks like for proteins to not be completely soluble. The proteins may clump together to form aggregates, but they may not. Either way, they are no longer able to dissolve into the water.