CPL Spectrum Measurement of Green Fluorescent Protein (GFP)

Download PDF February 12, 2018

Introduction

Circularly polarized luminescence
CPL-300 Circularly Polarized Luminescence Spectrophotometer

Circularly polarized luminescence (CPL) measures the difference in the emission intensities of left- and right-handed circularly polarized light of chiral compounds. Unlike CD, CPL provides information on the excited state properties of chiral molecules.

Green fluorescence protein (GFP) is a protein that emits green fluorescence when exposed to ultraviolet light and has been shown to have chiroptical properties1. This application note illustrates the CPL and CD spectra of GFP obtained with the CPL-300 and J-1500, respectively.

Experimental

Measurement Conditions
CPL-300 Circular Polarized Luminescence
Excitation Wavelength399 nm
Excitation Bandwidth
12 nm
Data Interval
1 nm
Response
16 seconds
Emission Bandwidth
8 nm
Scan Speed
10 nm/min
Accumulations
36
Path length
10 mm
Measurement Conditions
J-1500 Circular Dichroism Spectrophotometer
Data Pitch0.2 nm
D.I.T.1 sec
Bandwidth1 nmScan Speed200 nm/min
Accumulations16Path Length20 mm

Keywords

J-1500, Circular dichroism, CPL-300, Circularly polarized luminescence, Protein structure, Fluorescence, Biochemistry

Results

CD and absorption spectra (Figure 1, green) of a 0.03 mg/mL GFP solution was measured in the near-UV and visible regions. The GFP chromophore is observed in the visible region at 400 nm and the absorption bands due to the aromatic amino acid residues are observed in the near-UV region between 250 and 300 nm.  The CPL and fluorescence spectra were also obtained and show in blue in Figure 1. The characteristic fluorescent and CPL spectra of GFP are observed between 470-570 nm.

Figure 1. CPL (blue) and CD (green) spectra (top) of green fluorescent protein. The absorption (green) and fluorescence (blue) spectra are shown at the bottom.

References

1. Hiromasa Goto, Isao Sawada, and Nobuhiko Nomura, International Journal of Polymeric Materials, 2010, 59, 786-792.

About the Author

Leah Pandiscia received her PhD from Drexel University where she studied Biophysical Chemistry. She is a Spectroscopy Applications Scientist at JASCO.