CPL spectrum measurement of Green Fluorescent Protein (GFP)

Introduction

Circularly Polarized Luminescence (CPL) is the difference of circular polarized fluorescence intensity between right-handed and left-handed which is emitted from chiral compounds under exited condition. There are several main target samples and applications in CPL measurement as fluorescent chiral organic compound and chiral lanthanoid complex. And Biomacromolecule such as Green Fluorescent Protein (GFP) is also different target sample. GFP is a fluorescent protein which isolated from Aequorea victoria, the CPL spectra of the protein have already been reported, which indicates that living jelly fish may emit the circular polarized light ¹⁾. And Mantis shrimp is known to perceive circular polarized light and this detail mechanism is expected to be elucidated in future related with the research progress.

This application note shows CPL measurement results about GFP fluorescence protein by using CPL-300.

Experimental

[CPL-300: Circular polarized light luminescent measurement system]

CPL-300 Circular polarized light luminescent measurement system

Excitation wavelength: 399 nm
Measurement Range: 450 – 570 nm
Excitation bandwidth: 12 nm
Fluorescence bandwidth: 8 nm
Scan speed: 10 nm/min
D.I.T. : 16 sec
Data acquisition interval: 1 nm
Accumulation: 36 times
Optical Path Length: 10 mm

[J-1500: Circular dichroism spectrometer]

J-1500 Circular dichroism spectrometer

Measurement Range: 235 – 570 nm
Bandwidth: 1 nm
Scan speed: 200 nm/min
D.I.T. : 1 sec
Data acquisition interval: 0.2 nm
Accumulation: 16 time
Optical Path Length: 20 mm

Keywords

CPL, Green Fluorescent Protein, Biomolecule

Results

CD and absorption spectra of GFP solution (0.03 mg/mL) was measured in the wavelength range from
near UV to visible. The band of GFP fluorescent chromophore is observed in visible region. Additionally, in the near-UV region, the band of side chain of amino-acid aromatic is observed. (Fig.1, green line) And then, CPL and fluorescence spectra of the solution are measured using CPL-300. Characteristic fluorescent and CPL spectra of GFP are observed in the wavelength range from 470 nm to 570 nm.(Fig.1, blue line)