Even in fission yeast, RNA polymerase complexes can be critical for splicing

absence and presence of inhibitor, is the inhibition constant and is the concentration of inhibitor. 2.3. ThT fluorescence spectroscopic measurements The ThT fluorescence measurements were recorded on Shimadzu 5301PC fluorescence spectrophotometer equipped with water circulator from 460 to 600 nm after excitation at 440 nm. The excitation and emission slit widths were set at 10 nm. Prior to ThT fluorescence measurements, aliquots were supplemented with ThT and incubated for 30 minutes in dark. The obtained data was fitted against sigmoidal curve using equation in sigma plot. F Fi mi t Ff mf t 1 e tt0 t 2 where F is the fluorescence intensity at time t, and t0 is the time to attain 50% of maximal fluorescence intensity. and represent the initial base line related to the induction time and finnal constant line, respectively. The apparent rate constant for fibril growth is given by 1/, and the lag time is calculated by PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19729642 to -2. 2.4. Fluorescence microscopic measurements The aliquots were supplemented with ThT in 1:1 molar ratio of TPGS biological activity protein to ThT and incubated for 30 minutes in dark. The samples were washed thoroughly and visualized under fluorescence microscope using a 100X oil immersion objective. 2.5. Transmission electron microscopy TEM micrographs of HEWL in absence and presence of DCS and PYZ were taken on Philips CM-10 transmission electron microscope operating at an accelerating voltage of 200 kV. The samples on 200-mesh copper grid covered by carbon-stabilized formvar film. Excess of fluid was removed after 2 min and the grids were then negatively stained with 2% uranyl acetate. 2.6. Far-UV circular dichroism measurements Far-UV CD measurements were performed on a JASCO spectropolarimeter with a thermostatically controlled cell holder attached to a Peltier with multitech water circulator. Far-UV CD spectra were scanned in the range of 200250 nm in a cuvette of 0.1 cm path 3 / 21 Anti-Amyloid Behavior of Pyrazinamide and D-Cycloserine length. Each spectrum was an average of three scans. The percent secondary structure was calculated by K2D3 software. 2.7. Congo red binding measurements Increase in absorbance of Congo red along with red shift on protein interaction, is characteristic feature of amyloid formation.Aliquots incubated in absence and presence of drugs were supplemented with Congo red at a molar ratio of 1:1 with protein and incubated for 15 minutes. The absorbance spectra of the samples were recorded with a UV-visible spectrophotometer in a 1 cm path length cuvette. 2.8. ANS fluorescence measurements The ANS fluorescence measurements of aliquots incubated in absence and presence of drugs were performed on Shimadzu 5301PC fluorescence spectrophotometer equipped with water circulator. The excitation wavelength for ANS fluorescence was set at 380 nm and the emission spectra were recorded from 400 to 600 nm. Both excitation and emission slits were set at 5 nm. Prior to measurements, aliquots were incubated with 50 fold molar excess of ANS for 30 min in dark. 2.9. Steady state fluorescence quenching measurements All fluorescence measurements PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19728371 were done usingShimadzu 5301PC fluorescence spectrophotometer equipped with water circulator at 25C. The titration of the drugs to 5 M HEWL solutions was carried out in a dual-path length fluorescence cuvette. Intrinsic fluorescence was measured by exciting protein at 295 nm and emission spectra were recorded in the range of 300450 nm. The excitation and emission slits width were set at 3 nm