School of Engineering and Science - Jacobs University

Results
XAD4-purification yielded in a yellow-brownish mixture of substances; thus it
had to be further cleaned. The following three gel-filtrations using different
materials (Fractogel TSK HW 50s, Merck; Bio-Gel P-30, Biorad; and G25
Sephadex, Amersham) were conducted, resulting in an almost colorless
sample. Interestingly, upon addition of iron the color of the sample changed to
yellow. This is remarkable, as the D. chrysanthemi achromobactin was named
so because the iron-siderophore complex did stay colorless (Münzinger et al.,
2000). Unfortunately, the sample obtained after gel-filtration still contained
interfering substances and the resulting siderophore concentration was rather
low. Thus it was not suitable for NMR (Prof. Budzikiewicz, cologne, personal
communication). Therefore other methods were tested. HPLC separation on a
C18-RP sherisorp column did result in separation of several substances, but no
siderophore activity could be determined after the HPLC run (data not shown).
Next, separation of the sample by thin layer chromatography (TLC) was
conducted. Compared to column chromatography, TLC allows a rather fast
optimization of separation conditions, as it circumvents collection and testing of
numerous fractions per separation step. Theoretical models on the separation
characteristics of mobile phase mixtures allow a targeted modification of
solvent mixtures (Nyiredy, 2004; Snyder, 1978). After successful optimization of
the separation strategy, preparative TLC can be utilized or the running
conditions can be transferred back to column chromatography. TLC separation
of XAD4-purified supernatant of Pss22d∆Pvd was performed on silica plates
(Macherey-Nagel) in combination with various mobile phases. Separation of the
sample was investigated by autofluorescence of the substances at an excitation
wavelength of λ=360nm or quenching of a fluorescence indicator within the
silica matrix (excitation λ=254nm). The position of the achromobactin-like
siderophore was determined by CAS-overlay. A first TLC-separation of
Pss22d∆Pvd XAD4-purification using an equivolurimetric mixture of
diethylether, n-butanole, acetic acid, and water resulted in the separation of
several fluorescent substances (Fig.18).
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