drug-protein bound by hydrophobic interactions

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mariu
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drug-protein bound by hydrophobic interactions

Hello:
I have a brain homogenate in 1% triton and 150mM NaCl in PBS, in that homogenate I have a drug bound by hydrophobic interactions to my protein of interest.
How can I do to unbind them?
Thanks in advance

Neznika
Neznika's picture
spin homog ~15K g at 4degC

spin homog ~15K g at 4degC/30min twice -> take supernatant -> precipitate proteins with cold AmSO4 (1V/ 1V at slow stir at 4degC) -> spin ~15K g at 4degC/30min ->  wash pellet with 90% Acetone (1V pellet/ 3V Ac) -> dry benchtop at RT -> resuspend in buffer. Could help in your case

mariu
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Thanks Neznika:

Thanks Neznika:
I have read that the AmSO4 is used for crystalization..... then it shouldn't affect the native state of my protein, right?
I was also thinking about a more physical way, but my knowledge is kind of weak in that area. Could I try to break the hydrophobic interaction by incubating my homogenate with ionic beads? In theory they would be able to compete with the drug and displace it if I chose the right charge, is this correct?

Neznika
Neznika's picture
...AmSO4 is used for

...AmSO4 is used for crystalization.. - Yes. AmSO4 is used for the precipitation of proteins too.
...it shouldn't affect the native state... - Yes it'll not.
...my knowledge is kind of weak... - Don’t worry:)
...homogenate with ionic beadsI....- it’s possible. Use the gradient of 0-1.5M NaCl. NaCl decreases hydrophobic interactions. The protein could stick to beads, NaCl will decrease HI adn drug will go though column. Another way is to use a solvent.
...they would be able to compete with the drug... - I guess no because ionic beads interact with proteins according ionic interactions - charge-charge. The hydrophobic are based on non-charged interactions.

Sami Tuomivaara
Sami Tuomivaara's picture
Mariu and Neznika,

Mariu and Neznika,

If the drug-protein interaction is hydrophobic, using polar or charged buffers (NaCl, or Na+ and Cl- ions) or ionic beads makes the interaction even (relatively) stronger... So they are not an option.

I think your best bet is to use a mix of aqueous and organic solvents that denature the proteins and hence releases the drug, but where your drug is still soluble. Then you can spin down the protein pellet and recover the drug from the supernatant.

Cheers,

mariu
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The thing is that I need to

The thing is that I need to maintain my protein intact, so unfortunately denaturing is also not an option. As I have 150mM NaCl in my homogenate, would it help then getting rid of it by lets say dialysis?
If that helps breaking the interaction I would be able to recover my protein just by high centrifugation, because it is insoluble. My problem until now is that the drug was pelleted with the protein in my normal buffer, and that makes the protein unuseful for further experiments like cell assays because the drug is toxic.

Neznika
Neznika's picture
to Suola

to Suola
Yes. You are right about NaCl. Thanks
to Mariu
Sorry about NaCl. My fault. By the way what about the hydrophobic column?