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I'm attempting a derivatization method for polyamine detection using 4-fluoro-3-nitrobenzotrifluoride (FNBT) (Spragg and Hutchings, 1983). The published protocol tells you to quench the reaction with 1 M histidine in 1 M sodium bicarbonate. At first I thought this must be a typo because it's so far above the nominal solubility limit for histidine (it's also right at the limit for sodium bicarb) but I've seen multiple papers from different groups using this exact protocol.
So I'm wondering: how can I make this solution? I've tried sonication (water bath), upping the temp to 60 C, raising and lowering the pH, but no dice. I'm always left with a huge pile of histidine at the bottom of the tube. Note that I've only tried these manipulations over relatively short timescales (10-30 minutes), so it's possible that they might work if done overnight, but I'm not exactly optimistic. Any suggestions would be greatly appreciated.
Thanks for the suggestions. This is the relevant methods paragraph from the most recent paper to use this protocol (every other paper using the same method reads pretty much verbatim):
"The dried tissue was dissolved in 100 μl of 1 M sodium bicarbonate then derived with 300 μl of 4-fluoro-3-nitrobenzo-trifluoride (FNBT) reagent (a mixture of 10 μl of FNBT and 1 ml of dimethyl sulfoxide) at 60 °C for 20 min. At the end of derivation, 40 μl of 1 M histidine in 1 M sodium bicarbonate was added to the reaction mixture then the derivation continued for another 5 min to scavenge excess FNBT. After cooling the mixture in an ice basket, the N-2′-nitro-4′-trifluoromethylphenyl (NTP) derivatives of polyamines were extracted twice with 2 ml of 2-methylbutane."
There's nothing there that suggests to me that the histidine is dissolved in anything but water. Same for the original derivation paper...
I could try using a histidine salt (all I had available at the time was pure) but it doesn't seem likely to get the rest into solution, especially given the fact that I'm working in a solution already saturated with sodium bicarbonate.
See, to me, this would suggest that the histidine/bicarb might be dissolved in DMSO. After all, your FNBT reagent is in DMSO.
If nothing else, histidine/bicarb dissolved in DMSO would be miscible with your FNBT/1M bicarb solution, and because you are only adding 40uL to 400uL of existing solution, at least some of which is DMSO solvent, I don't think it will matter much chemistry-wise whether the histidine/bicarb is dissolved in DMSO or water. (Well, besides the fact that you can't dissolve that much histidine in water.) But try the histidine salt because this seems like a good suggestion.
I'm sure the hydrochloride will be more soluble than the free amino acid. Whether it will be soluble to the tune of 1 M or not, I can't say. I know you can get things like Arg in solution at 0.5 M and I'm pretty sure it's the hydrochloride. It sounds strange to me to make up a salt (sodium bicarbonate) in DMSO, but hey, if it works...The FNBT is not going to be very water soluble, so that doesn't surprise me that you'd want to have your stock solution of FNBT, at least, in something like DMSO or ethanol.
7 posts • Page 1 of 1
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