Dear Researchgate Community,
I am currently trying to couple taurine to a protein via EDC reaction. I have already tried the following attempts:
1) Direct coupling at either pH 5 - 6, by only using EDC
2) Direct coupling in a two-step reaction with the aid of NHS
3) Succinylation of the lysine residues of the protein, then coupling by EDC only at pH 6
For all attempts, I have used around 7-fold molar excess of EDC to expected -COOH groups of the protein, and around 9-fold molar excess of taurine, if not even up to 20-fold.
The way I analyze "coupling efficiency" is by measuring the zeta potential of the dialyzed product at different pH, in hopes that since taurine "masks" lysine residues and offers a sulfonic acid instead, the IEP must drift to more acidic pH values. After succinylation alone, such a drift could be observed, but after EDC reaction, I keep finding an IEP that is been even more basic than my native protein, which could be caused by side-reactions due to EDC. After EDC/NHS reaction, the IEP is unchanged compared to the native protein.
I have found a paper (doi: 10.1007/bf01046841) in which taurine was succesfully coupled to succinylated ovalbumin and couldn't find anything that I did differently in terms of pH and amounts of reagents. My only "weak points" would be the total volume I'm working with. Judging by this, I have always used more concentrated solutions of protein, taurine and EDC (by using less water). I wouldn't expect this to impact the reaction, but I might be very mistaken. Does anyone have experience or knowledge about this?
I would be very much thankful for any help. Could it really be the reaction volumes? Am I using too much EDC, facilitating the reaction to N-acyl-urea derivates (although I have found similar amounts of EDC in literature, and have used the same amount to successfully couple ethylenediamine to the same protein before)? Am I overlooking something vital?
Thank you in advance.
One possible issue could be the amount of water in your reaction. Using more concentrated solutions of protein, taurine, and EDC means that there is less water in the reaction, which can increase the chances of side reactions occurring. Additionally, if the reaction mixture is too concentrated, it may be more difficult for the reagents to diffuse to the protein surface and react efficiently. Therefore, you may want to consider increasing the total reaction volume and using more dilute solutions of your reagents.
Another factor to consider is the pH of the reaction. You mentioned that you have tried coupling at pH 5-6 and pH 6, but it may be worth trying a wider range of pH values to see if this affects the coupling efficiency. Additionally, you may want to consider optimizing the pH of the reaction for the specific chemistry involved in taurine coupling. For example, the optimal pH for EDC/NHS coupling may be different than the optimal pH for direct EDC coupling.
Finally, you may want to consider modifying your EDC reaction conditions to minimize the formation of N-acyl-urea derivatives. This could include using a lower molar excess of EDC, optimizing the reaction time, or using different reaction conditions altogether.
Overall, it may be helpful to consult with a protein chemistry expert or experienced colleague to troubleshoot the specific issues you are encountering. They may be able to provide additional insights or suggest alternative approaches to achieve your desired coupling efficiency.
Thank you Mr. Guobing Xiang for your suggestion and linked discussion!
Thank you Mr. Ahmad Al Khraisat for your detailed answer and explanations!
I will retake my approaches with diluted solutions and less EDC, maybe adding little amounts of EDC after a certain period of time to the reaction, instead of using all at once.
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