EK FL 3 #37

[sfsn]

My question is about how to choose the solvent (from among mercaptoethanol, urea, or GuHCl) to create a favorable environment for activity of a hydrophobic enzyme. I think I'm not understanding this question because I don't really get how these solvents would (or would not) interact with something hydrophobic.

1. I'm trying to understand this in terms of like-dissolves-like - is this how I should be thinking about this?

2. Since beta-galactosidase is mostly hydrophobic, do we want to use urea because it is NOT hydrophobic?

3. The explanation says urea acts primarily on hydrogen bonds and mercaptoethanol acts mainly on disulfide bonds. So urea would be less likely to interact with hydrophobic beta-galactosidase because...why?

4. And why is urea a better solvent choice than mercaptoethanol?

5. Why would GuHCl "most disrupt hydrophobic interactions"?

6. I read in some abstract that turned up in my search that urea and GuHCl are non-polar. But it looks polar. So which is it?

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[salemstein]

1) Yes
2) Yes
3 and 4) mercapethanol, though hydrophilic, disrupts tertiary structure (it kills the S-S bonds), so you shouldn't use it to keep protein intact.
5) "Organic solvent" usually means hydrophobic stuff blends well with it. Think "organic layer" vs "aqueous layer" in orgo separation
6) Not sure about this one, but Guanidine in acid is non-polar (because of resonance associated with protonated N+?). Urea is polar.

 

[aldol16]

You should know these common denaturing agents. You should know what each does and how it does it.

Beta-mercaptoethanol is a reducing agent. It will reduce disulfide bonds. Many protein tertiary structures and/or monomeric units are linked together by disulfide bonds and beta-mercaptoethanol will trash that.

Urea is a denaturing agent. Look up the structure of urea. It's got a lot of hydrogen bond donors. So it works by providing hydrogen bonds to the unfolded form of the protein, i.e. the backbone amides/carbonyls, polar side chains, etc. If you want the protein to stay folded, you want a low concentration of urea. 1 M is generally low because in denaturing studies, you usually use 8 M urea to denature everything.

Guanidinium HCl is a salt that provides both hydrogen bonding and ionic contacts to stabilize the unfolded protein.

1) Yes
2) Yes
3 and 4) mercapethanol, though hydrophilic, disrupts tertiary structure (it kills the S-S bonds), so you shouldn't use it to keep protein intact.
5) "Organic solvent" usually means hydrophobic stuff blends well with it. Think "organic layer" vs "aqueous layer" in orgo separation
6) Not sure about this one, but Guanidine in acid is non-polar (because of resonance associated with protonated N+?). Urea is polar.

1) Yes, with the caveat that it's not dissolving anything. Here, you're interested in stabilization of the unfolded form vs. the folded form.

2) Urea is a common denaturing agent. The key is that you're using a low amount of it here. If you want to keep the protein intact, you would ideally use no urea but that's not an option here. Here's where the "like dissolves like" approach runs into trouble.

3 - 4) Again, it's the fact that you don't have the option of not using urea and a reducing agent is worse because it will destroy tertiary/quaternary structure if there are disulfide bonds in those.

5) GuHCl, like urea, provides H-bonding to the backbone amides/carbonyls, which would stabilize the unfolded form. In the folded form, it can't access most of the backbone because it's buried.

6) Urea is polar. Look at the structure. It's got a carbonyl group and the oxidation state of the carbon is equivalent to that of CO2. GuHCl, however, is relatively nonpolar for the same reason BF3 or AlCl3 is non-polar (hint: it's trigonal planar).

 

[theonlytycrane]

@aldol16 so between urea and the salt, it's the extra ionic interactions from the salt that disrupts the protein more than urea?

 

[aldol16]

@aldol16 so between urea and the salt, it's the extra ionic interactions from the salt that disrupts the protein more than urea?

I believe so, yeah. They're both commonly used as denaturing agents in protein biochemistry.

 

[NextStepTutor_2]

@aldol16 so between urea and the salt, it's the extra ionic interactions from the salt that disrupts the protein more than urea?

It would be good to know the relative (I use that term loosely as proteins have unique folding properties) but in general, you can expect Guanidine isothiocyanate > GuHCl > urea in terms of denaturing ability of an "average" protein at a given concentration. This is usually measured via equilibrium denaturation experiments and recording the concentration of the agent at the mid-point of denaturation. The lower this value, the stronger the denaturing agent.

Antoher important property is to note that GuHCl is charged while urea is neutral.

GuHCl

Urea


This has an effect on the stability of a protein above and beyond its effect on water structure and its competition for H-bonds. The net charge of the protein and it's pH stability will relate to Gu unfolding but not so much to urea unfolding. That is, if the protein is highly charged, with stabilizing salt bridges, then it will be more destabilized by GuHCl than by the same amount of urea.

protip: my biochem friends tell me if you are looking for even better denaturant than GuHCl then use GuSCN, it is better and more effective.

Hope this helps, good luck!

 

[aldol16]

This has an effect on the stability of a protein above and beyond its effect on water structure and its competition for H-bonds. The net charge of the protein and it's pH stability will relate to Gu unfolding but not so much to urea unfolding. That is, if the protein is highly charged, with stabilizing salt bridges, then it will be more destabilized by GuHCl than by the same amount of urea.

To make this clearer, the folded form of the protein will be destabilized by the GuHCl whereas the unfolded form would be stabilized by GuHCl, thus driving the equilibrium F ---> UF to the right.