Is it ok for your system to change the pH

This is a really good point.

So my solution has a pH around 7.5 and the CAS assay I perform uses PIPES as buffer which works till 7.5.

So far I didn´t try to change (decrease) the pH because:

1) the CAS assay could not work for really acid pH

2) I don´t know if I will demage the molucules

3)if I decrease the pH I could detach the iron due to the protonation of the residues in the siderophore but at the same time the Iron will be still in the solution, how I can remove it then?

Do you have any suggestion cencerning pH variation?

Thank you very much for your help!

To remove iron from the siderophore, you may consider lowering the pH (as Dr. Bashir suggested) then pass the solution through a Chelex column. Chelex (iminodiacetic acid) has high affinity for multiple different metals. The acid itself is immobilized on beads so the chelator itself won't be introduced into your solution. Collect the flow-through from the column and (hopefully) it should contain your iron-poor siderophore. Normalize the pH to 7.5 and you should be okay. Hope this helps

There may be many options, first you can directly measure your siderophore (even boudn to iron) through LC MS, if you do not have access or do not want to sue that then one wild idea may be lower the pH temporarily then purify the siderophore through column and then measure

Thank you very much for your help!

In the first istance I don´t really know the features of my chelating agent. Meaning I don´t really know how big it is and how it looks like. It is produced by my bacterial strain during growth and I´m doing all my test with the spent medium of the culture. Therefore in solution I have several compounds. I tested my solution with an LC and I obtained some fraction wich contains iron, but since my sample is a complex mixture I cannot be sure that in those fraction I just have my chelating agent.

I already tryed an O/N Chelex treatment but only a small amoun of iron was removed.

I could actually try to lower the pH around 3 and treat with Chelex. I´m afraid that more acid condition could compromise the structure of my molecule.

I will be really happy to hear your further suggestions.

I would try lowering the pH then treating with Chelex. You are right that acidic pH may damage the siderophore but without knowing its identity you can't be sure. Do you know how stable other bacterial siderophores are over a range of pH values? If others are stable at pH 3, it's likely that yours might be too.

First of all cas media preparation vary pro cation

Beware the pH. When we try to use a slightly acidic pH of the mobile phase in HPLC (CH3CN/H2O mixtures 0.1% TFA) in the isolation of siderophore piscibactin , the compound decomposed. We had to isolate in chelated form as gallium complex and never use an acidic pH.

Dr Jimenez, this is a really interesting news to know!

Thank you so much!

Siderophore production is up-regulated under iron limitation.

Are your cultures iron limited?

Are you growing cultures on Fe-free media (ideally you should adhere to trace metal clean protocols) ? This should increase the signal significantly. If the signals are low , try growing cultures to high biomass under iron rich conditions and then transferring them (after a good wash with EDTA medium and then Fe-free meium) to Fe-free media. If signals are still low- you can freeze dry the solution and then reconstitute in a smaller volume of MeOH.

Good Luck,

Hagar

Extract the siderophores by the standard methods you have been using without adding any iron, so the siderophore obtained will be in a deferrated form, provided there is no Fe in the growth medium.