Re: Gramicidin purification
Dan Brune (DBrune@asu.edu)
Thu, 08 Oct 1998 10:27:23 -0700 (MST)
Dear Laurent,
I do not really have an answer to your question about purifying
Gramicidin D, but have a few comments that I hope may be helpful.
Yesterday, a student brought over an "aqueous solution" of that protein for
analysis by mass spec. After not getting any reasonable mass spectrum, I
found a Merck Index and noticed that gramicidin D is nearly insoluble in
water (about 0.6 mg/100 ml, if I remember correctly). It is soluble in
"lower" alcohols and moderately soluble in dry acetone and dioxane. One
possible problem with your purification might be that gramicidin is just
not soluble in the usual acetonitrile water mixtures (or methanol water
mixtures) used in reversed phase HPLC. Because gramicidin D has several
tryptophans, it should have a fairly high uv extinction coefficient so that
its concentration in solution can be measured easily. I also noticed that
the Fluka catalog states that their gramicidins A and C, which are similar
to D, are at least 90% pure by HPLC, so purification by HPLC should not be
a hopeless task. Perhaps it can be eluted using a less polar alcohol such
as propanol, or even a mixture of propanol and a less polar solvent like
hexane. I also noticed in the Fluka catalog that gramicidins A and C (and
by inference, also D) lack basic amino acids (also they lack both the
N-terminal amino group and the C-terminal carboxyl groups), so I would
guess that there is no need to include TFA in the solvent system as an ion
pairing agent.
Good luck with the purification.
Dan Brune
>Hello everybody,
>I need to purify some Gramicidin D (from SIGMA no G-5002) : I would like
>to separate the different isoforms by RPHPLC (with a C4 column).
>I already tried with ACN/H2O and MeOH/H2O as mobile phase but
>unsuccessfully in both cases : No signal after column !! the peptide's
>maybe stuck into my column (?).
>Any suggestions (eluents, gradients, sample solvent...)?
>
>Thanking you in advance for your help,
>LAurent.