production of selected secondary metabolites in transformed ...

microenvironment polarity is well known and described (Aguiar 2003). We evaluated
experimental data using non-linear fitting with Boltzman’s curve with four parameters –
maximum (a), minimum (b), inflex point (x0), and width of the gradient (Δx) (Equation 1).
We voted and subsequently confirmed the “x-coordinate” of the inflex point as the cac value.
For the confirmation we used the perylene’s fluorescence measurements.
a − b
y = + b
1)
( 0
x − x )
Δx
1+
e
Perylene, dibenz[de,kl]anthracene, is also the even and alternating hydrocarbon (Figure
2b). The perylene’s measurements are quite simple for evaluation. The fluorescence intensity
of the perylene rise with the number of non-polar domains in the hHA’s solution. Earlier than
domains are presented in solution no fluorescence is observed. When domains are formed we
observe sharp increasing of the fluorescence. These two trends can be fitted by the linear
curves and the x-coordinate from their point of intersection defines the cac value directly.
a) b)
Figure 2 Fluorescence probes - a) pyrene b) perylene
MATERIALS AND METHOD
The hyaluronan and its derivatives were obtained from CPN Ltd. (Dolní Dobrouč, Czech
Republic). Hyaluronans were in these molecular weights: 97, 560, and 1630 kg·mol -1 .
Derivatives were in molecular weights 134, 183, 360, and 1470 kg·mol -1 , respectively, and
theirs substitution degrees were in range from 10 to 70 %. The substitution degree is defined
as the ratio of the number of the monomer with and without the alkyl chain per polymer
chain, and it was determined from 1 H NMR spectra. All molecular weights were determined
by SEC-MALLS (Mlčochová, 2006). Pyrene and perylene were obtained both from Fluka
GmbH. Acetone p.a. was obtained from Lachema Ltd.
The samples were dissolved in doubly distilled water to the concentration 2 g l -1 . This
stock solution was stabilized by addition sodium azide (NaN3) in final concentration 10 -3 M.
Sample nomenclature. The samples are named in correspondence to their characteristics. The
first come alkyl-type abbreviation, next are basic molecular weight (before derivatization),
and after the solidus the substitution degree. For example D 134/10 means C10-derivate with
the molecular weight 134 kg·mol -1 and the substitution degree 10 %.
Fluorescence Method. The acetone stock solutions of the pyrene and perylene were prepared.
Probes stock solution was introduced into a flask and acetone was evaporated. The stock
solution of hHA was introduced into a flask with evaporating probe, it was diluted to the
desirable concentration, and the resulting solution was sonificated during 4 hours and stored
during next 20 hours. The fluorescence emission spectra were monitored with a luminiscence
Sborník soutěže Studentské tvůrčí činnosti Student 2006 a doktorské soutěže O cenu děkana 2005 a 2006
Sekce DSP 2006, strana 214