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Introduction

The intrinsic fluorescence of proteins is due to the aromatic amino acids tryptophan, tyrosine, and phenylalanine. Tryptophan, which excites maximally around 270-280 nm and has an emission peak near 350 nm in water, dominates the emission of proteins and is the most sensitive to solvent polarity and the local environment. Exposure of tryptophan residues to water, which occurs when a protein is denatured, leads to a shift to longer emission wavelengths. This shift in peak emission can be used to monitor protein unfolding.

In this application protocol we demonstrate performance of the SpectraMax® i3 Multi-Mode Microplate Platform for assays measuring intrinsic tryptophan fluorescence. High sensitivity is demonstrated with a tryptophan standard curve, and a shift in peak emission is shown using a lysozyme denaturation assay. Lysozyme contains two tryptophan residues that fluoresce when excited with UV light. When lysozyme is denatured, tryptophan’s emission peak typically shifts about 6-10 nm. This peak shift can be measured by performing fluorescence spectral scans on native and denatured lysozyme.

The SpectraMax i3 Platform offers the sensitivity and wavelength scanning capability needed to perform intrinsic tryptophan fluorescence measurements. Additionally, SoftMax® Pro Software can automatically identify emission peaks and calculate the peak shift, streamlining analysis.

Materials and Methods

• SpectraMax i3 Multi-Mode Microplate Reader
• L-Tryptophan (Sigma P/N T-0254)
• Lysozyme (Sigma P/N L6876)
• Guanidine hydrochloride (Sigma P/N G7294)
• Black-walled, UV-clear 96-well microplates (Greiner P/N 655809)
• Phosphate buffered saline (PBS), pH 7.4

Standard curve

A standard solution of 1 mM tryptophan was prepared in PBS and diluted serially 1:2 down to 7.8 nM. Standards were pipetted in quadruplicate into a 96-well black-wall, UV clear-bottom plate at 200 µL per well. Eight wells containing PBS only were included as blanks for calculating the lower limit of detection.

Excitation and emission scans were performed on the SpectraMax i3 Reader to determine the wavelength pair giving optimal sensitivity. The lower limit of detection (LLD) was calculated based on 3x the standard deviation of the blank wells.

Protein denaturation assay

Guanidine hydrochloride at a final concentration of 5 M was used to denature a 10 mg/mL solution of lysozyme. Spectral scans were performed on native and denatured lysozyme samples using the SpectraMax i3 Multi-Mode Platform with excitation at 270 nm and emission ranging from 300 to 450 nm. Emission peaks were automatically identified using a preconfigured protocol in the SoftMax Pro Software.

Results

Using excitation at 270 nm and emission at 350 nm on the tuneable SpectraMax i3 Multi-Mode Platform, tryptophan could be detected down to about 6 nM (Figure 1). This level of sensitivity is more than sufficient to measure proteins in denaturation studies and other studies where a shift in emission peak occurs. Spectral scans of native and denatured lysozyme samples showed a shift in the emission peak from 351 nm to 361 nm, along with an increase in fluorescence intensity, with denaturation (Figure 2).

Using the optimal wavelength combination, the SpectraMax iD3 reader was able to accurately measure the tryptophan standard curve with a lower limit of detection at 9.5 nM and excellent linearity with r2 > 0.99 (Figure 2).

Figure 1. Tryptophan standard curve. Tryptophan was serially diluted in PBS to make a standard curve. Linearity and sensitivity down to 6 nM was demonstrated.

Figure 2. Peak shift of denatured lysozyme. Top: Emission spectral scans of native (red) and denatured (blue) lysozyme. A peak shift of 10 nm was observed. Bottom: SoftMax Pro Software group table showing automated analysis of peak shift in the preconfigured protocol.

Conclusion

The SpectraMax i3 Multi-Mode Plate Reader enables characterization of proteins by measuring intrinsic tryptophan fluorescence. Monochromator optics with adjustable bandwidth offer excellent sensitivity down to 6 nM tryptophan, as well as precise determination of the fluorescence emission peak shift that occurs upon protein denaturation. With a pre-configured SoftMax Pro Software protocol, peak wavelength and peak wavelength shift are identified automatically, saving time on analysis.

The SpectraMax® i3 Platform from Molecular Devices is a multi-mode detection system that offers a broad range of application possibilities. Monochromator optics support absorbance, fluorescence, and luminescence, while user-exchangeable cartridges expand the system’s detection to time-resolved fluorescence, fluorescence polarization, and AlphaScreen modes. The user-upgradeable SpectraMax® MiniMax™ Imaging Cytometer option makes this platform one of the most versatile microplate detection systems available.

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