Molecules 2019, 24, 1600
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3.2. Peptides 1–18 Analysis
Analytical RP-HPLC. Performed on a Waters HPLC system (Waters Corporation, Milford, MA,
USA), using a Kinetex Reversed Phase C18 column (100 4.6 mm). A gradient of 0.1% TFA in H2O (B)
and 0.1% TFA in CH3CN (A), at a flow rate 0.4 mL/min was used with UV detection at 220 and 254 nm.
All HPLC spectra of crude 18 peptides are available in Supplementary Materials: Figures S1, S3, S5,
×
1–
S7, S9, S11, S13, S15, S17, S19, S21, S23, S25, S27, S29, S31, S33 and S35.
MS analysis. Performed on MS Bruker microOTOF-QIII (Bruker Corporation, Billerica, MA, USA).
MS spectra of 1–18 peptides are available in Supplementary Materials: Figures S2, S4, S6, S8, S10, S12,
S14, S16, S18, S20, S22, S24, S26, S28, S30, S32, S34 and S36.
Preparative HPLC. Performed on a CombiFlash, EZPrep, Teledyne ISCO (Lincoln, Nebraska,
USA) using a Supelco Discovery BIO Wide Pore C18 column (25 cm
×
21.2 mm, 10 mm; Sigma-Aldrich);
flow rate, 5 mL/min; detection wavelengths, 220 and 254 nm) with gradient ratio A (0.1% TFA in
MeCN) and B (0.1% TFA in H2O) 0:100 to 18:82 in 30 min, followed by an isocratic run for 5 min.
Spectroscopic measurements. Performed on UV spectrophotometer Hitachi (Hitachi, Tokyo,
Japan), in a wavelength range from 400 nm to 800 nm. UV spectra of
Supplementary Materials: Figures S37–S54.
1–18 peptides are presented in
Fluorescence measurements. Performed on FLUOROMAX-3 Horiba Scientific (Edison, NJ, USA)
in a wavelength range from 470 nm to 600 nm, excitation wavelength 440 nm. Fluorescence intensity
spectra of 1–18 peptides are presented in Supplementary Materials: Figures S56–S73.
3.3. Spectroscopic Measurements with Congo Red (GP 5)
To initiate the aggregation process, peptide samples were incubated for 7 days at 37.4 ◦C in 1 mL of
phosphate buffer solution (concentration 0.1 M, pH 7.2), when difficulties in the solubility of peptides
in the buffer were observed, samples were sonicated for 15 s. The final concentration of the incubated
peptides was c = 1.44 mM. Subsequently, 1 mL of Congo Red (Sigma-Aldrich) solution (c = 45 µM,
phosphate buffer, pH 7.2) was added to samples, which were incubated for a further 4 days at room
temperature. During this period spectroscopic measurements were performed in the wavelength range
of 800 nm to 400 nm. Aggregation studies of all the incubated samples spectroscopic measurements
were begun 30 min after the addition of the Congo Red solution. Registered spectra of a mixture
containing 1 mL solution of Congo Red (c = 45
µM, phosphate buffer, pH 7.2) and of a 1 mL of solution
of phosphate buffer (concentration 0.1 M, pH 7.2), also incubated for 4 days at room temperature,
were used as controls. All UV–Vis spectra peptides
Supplementary Materials.
1–18 incubated with Congo Red are presented in
3.4. Spectroscopic Measurements with Thioflavin T (GP 6)
◦
To initiate the aggregation process, peptide samples were incubated for 7 days at 37.4 C in
2 mL of solution of phosphate buffer (concentration 0.1 M, pH 6.0), when difficulties in the solubility
of peptides in the buffer were observed, samples were sonicated for 15 s. To the samples was then
added 2 mL of Thioflavin T solution (c = 57 mM, phosphate buffer, pH 6.0). The samples were
incubated for another 4 days at room temperature. The final concentration of the incubated peptides
was c = 0.139 mM. Starting 30 min after the addition of Thioflavin T solution, over the following
days fluorescence measurements were performed in the wavelength range from 470 nm to 600 nm
(excitation
λ = 440 nm). Registered spectra of the mixture containing 2 mL solution of Thioflavin T
(c = 57 mM, phosphate buffer, pH 6.0) and 2 mL of phosphate buffer solution (concentration 0.1 M,
pH 6.0), also incubated for 4 days at room temperature, were used as the controls. All fluorescence
spectra peptides 1–18 incubated with Thioflavin T are presented in Supplementary Materials.