Intrinsic tryptophan fluorescence (ITF) emission spectra were recorded in a Hitachi F-2000 spectrofluorimeter at 25 °C using λexc = 295 nm (slit 10 nm), λem between 305 and 500 nm (slit 10 nm) and a scan speed of 240 nm·min−1. Samples contained 20 μg·mL−1 of protein (final concentration) to maintain A295 < 0.02. The l -Phe effect was monitored by incubating the protein samples with 1 mM l -Phe for 5 min, at 25 °C, prior to ITF analysis.
F 2000 spectrofluorimeter
Manufactured by Hitachi
Sourced in Japan
The F-2000 spectrofluorimeter is a laboratory instrument used for fluorescence analysis. It measures the intensity of fluorescent light emitted by a sample when it is exposed to light of a specific wavelength. The F-2000 can be used to identify and quantify fluorescent compounds in a variety of sample types.
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5 protocols using f 2000 spectrofluorimeter
1
Intrinsic Tryptophan Fluorescence Spectroscopy
2
Kinetics of Nitro-Naphthalimide Reactions
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The reactions of 4-nitro-NBN and 4-nitro-NEHN with thiols were followed at 400 nm in a Cary 3E spectrophotometer, in quartz cuvettes with 1.0 cm path length, using 2.5 mL total volume. All kinetics were performed at 30 °C. The buffers (0.01 M, except when indicated) used were: sodium acetate (pHs 5.0 and 5.5), MES (pHs 5.1; 5.3; 5.5; 6.0; 6.5 and 7.1), Tris/HCl (pH 7.0; 7.5), CAPSO, pH 10.6. Buffers were prepared with deionized water, saturated with N2 before use to minimize thiol oxidation during the kinetics. No TCEP was used in the kinetic studies because it reacts with nitro-naphthalimides. Fluorescence measurements were performed in a Hitachi F-2000 spectrofluorimeter at 700 V.
3
ROS Production Monitoring Techniques
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The production of ROS was monitored using different techniques and probes. The chemiluminescence of the Cypridina luciferin analog (CLA) react mainly with O2•− and 1O2 with light emission [22 (link)]. Chemiluminescence from CLA was monitored using a FB12-Berthold luminometer (with a signal integrating time of 0.2 s). For data analysis, the luminescence ratio (L/Lbasal) was calculated by dividing the luminescence intensities of CLA-luminescence (L) with the luminescence intensity before treatment (Lbasal). Hydroxy radicals (HO•) formation was also checked using the specific probe hydroxyphenyl fluorescein (HPF) [23 (link)]. Briefly, HPF was added to 1mL of MS medium to a final concentration of 10 µM at different times after the addition of 100 mg.mL−1 of CaPPs. The fluorescence increase was monitored at 515 nm after an excitation at 490 nm using a F-2000 spectrofluorimeter (Hitachi, Tokyo, Japan).
For biological production of ROS, we used the chemiluminescence of luminol [51 (link)], which is dependent on the activity of cell-derived peroxidase. Briefly, 6 mL of the cultured cells were inoculated with CaPPs. Before each measurement, 200 µL of the cell culture was added prior to the addition of 5 µL luminol (1.1 mM). Chemiluminescence measurements were carried out at 30 min intervals using a FB12-Berthold luminometer (signal integrating time 0.2 s).
For biological production of ROS, we used the chemiluminescence of luminol [51 (link)], which is dependent on the activity of cell-derived peroxidase. Briefly, 6 mL of the cultured cells were inoculated with CaPPs. Before each measurement, 200 µL of the cell culture was added prior to the addition of 5 µL luminol (1.1 mM). Chemiluminescence measurements were carried out at 30 min intervals using a FB12-Berthold luminometer (signal integrating time 0.2 s).
4
Quantitative Determination of Thiol Groups
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For quantitative determination of SH groups, both 4-nitro-NBN and 4-nitro-NEHN were used in excess. In a typical experiment, 10 μL of a 10 mM solution of naphthalimide (4-nitro-NBN in methanol and 4-NEHN in acetonitrile), to yield a final concentration of 250 μM of the probe, were added to 0.4 mL of buffer (in general Tris/HCl 0.01 M pH 7.5) containing CTAC 5 mM. To this solution a thiol aliquot was added at concentrations, at least, five times lower than the concentration of the naphthalimide. Fluorescence measurements were performed in a Hitachi F-2000 spectrofluorimeter at 700 V.
For quantitative determination of the SH contents of PRDX2, after stabilization of the fluorescence, the emission intensity was determined in the plate reader Fluorimeter using, for 4-nitro-NBN, λexc = 390 nm and λem = 463 nm and for 4-nitro-EHN, λexc = 400 nm and λem = 487 nm at 700 V. A plate reader Synergy H1 Hybrid Reader from Biotec was used to determine fluorescent intensity. All experiments were repeated three times, except for PRDX2 that was done induplicate.
n-Hexadecyl-cysteinamide chloride (HCys) and 4-Nitro-N-butyl-1,8- naphthalimide (4-nitro-NBN) were synthesized as described previously [15 , 20 ].
For quantitative determination of the SH contents of PRDX2, after stabilization of the fluorescence, the emission intensity was determined in the plate reader Fluorimeter using, for 4-nitro-NBN, λexc = 390 nm and λem = 463 nm and for 4-nitro-EHN, λexc = 400 nm and λem = 487 nm at 700 V. A plate reader Synergy H1 Hybrid Reader from Biotec was used to determine fluorescent intensity. All experiments were repeated three times, except for PRDX2 that was done induplicate.
n-Hexadecyl-cysteinamide chloride (HCys) and 4-Nitro-N-butyl-1,8- naphthalimide (4-nitro-NBN) were synthesized as described previously [15 , 20 ].
5
ROS Determination using DCFH-DA
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Determination of ROS was performed by using the fluorescent probe 2',7'-dichlorofluorescein diacetate, DCFH-DA, as previously described [39 (link)]. Briefly, 100 μL of 100 μM DCFH-DA, dissolved in 100% methanol, was added to the cellular medium, and cells were incubated at 37°C for 30 minutes. After incubation, cells were lysed and centrifuged at 10,000 x g for 10 min. The fluorescence (corresponding to oxidized CDF) was monitored spectrofluorometrically (λex = 488 nm; λem = 525 nm), using an F-2000 spectrofluorimeter (Hitachi) and results were expressed as Fluorescence Intensity (F.I.) x 1 x106/mg protein. Total protein content in each sample was evaluated according to Lowry et al. [40 (link)].
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