Fp 6300 spectrofluorimeter

The aggregation kinetic of free of PEGylated human amylin (10 mM buffer, 20 μM ThT, 50 μM peptide = 0.2 mg/mL, with 2% DMSO final concentration,) was performed in either continuous measurement in a multiwell plate (Corning Costar black plate; in a SpectraMax M5 spectrofluorimeter) or in separated samples (by using a Jasco FP6300 spectrofluorimeter). In this case, at the indicated time 160 μL sample were mixed with 40 μL ThT 100 μM. In both cases the thioflavin T fluorescence was measured by setting excitation at 450 nm and emission at 482 nm. The presence of residual (2%) DMSO shows no significant effect on human amylin aggregation as reported elsewhere [64 (link)] and in our own control experiments performed with varying concentration of DMSO (S1 Fig), demonstrated no significant contribution up to about 6% DMSO. The aggregation curves were fitted using a 4 parameter logistic function and the t_1/2, elongation rate and lag time were estimated as described elsewhere [65 (link)].

Fluorescence emission spectra were obtained using the method of Li et al. [10] (link). Briefly, 25 µL of 1 U/mL ACE (prepared in 50 mM Tris–HCl buffer, pH 7.5, containing 300 mM NaCl) or 25 µL of 250 µg/mL renin (prepared in 50 mM Tris–HCl buffer, pH 8.0, containing 100 mM NaCl) was mixed with 50 µL of peptide solution and 25 µL of the appropriate buffer to yield a 100 µL assay solution. Emission spectra were recorded at 25°C using a micro quartz cell (100 µL capacity) on a JASCO FP-6300 spectrofluorimeter (JASCO, Tokyo, Japan). ACE or renin assay solutions were excited at 280 nm and emission recorded from 290 to 450 nm. Emission spectrum of the respective buffer, peptides and enzymes was subtracted from the emission spectrum of each assay solution.

Purified pyrene-labelled rabbit G-actin, (Cytoskeleton Inc.) at 0.6 pyrene/monomer equivalence, was prepared at 2.5 μM in 5 mM Tris–HCl pH 8.0, 0.2 mM CaCl₂. An equal volume of aldolase, CTD, or bovine serum albumin (up to 50 μM final concentration) or no protein was then added prior to polymerization. Actin polymerization was initiated by transfer at a 1 in 10 ratio to 500 mM KCl, 20 mM MgCl₂, 10 mM ATP according to the manufacturer's instructions, in 10 mm QS cuvettes (Hellma GmbH & Co. KG). Fluorescence measurements were performed in a JASCO FP-6300 spectrofluorimeter at 20 °C and the fluorescence emission intensity recorded for 10 min using an excitation wavelength of 366 nm and an emission wavelength of 407 nm as described previously for pyrene-labelled actin [33] (link). The rate of actin polymerization was measured as the increase in fluorescence intensity per min. All reactions were carried out in duplicate.

The method described by Ijarotimi et al. [42 (link)] was used to record intrinsic fluorescence spectra on the FP-6300 spectrofluorimeter (Jasco Corp., Tokyo, Japan) at 25 °C with a 1 cm path length cuvette. Protein stock solution (10 mg/mL) was prepared in 0.1 M sodium phosphate buffer (pH 3.0, 5.0, 7.0, and 9.0), followed by centrifugation and the determination of the protein content of the supernatant. The supernatant was then diluted to 0.002% (w/v) and fluorescence spectra were recorded at an excitation wavelength of 275 nm (tyrosine and tryptophan) with emission recorded from 280 to 450 nm. The emission of the buffer was subtracted from that of the respective samples to obtain the reported fluorescence intensity (FI) spectra.