Skip to content

JASCO JASCO

  • News
  • Events
  • E-Store
  • My Account
  • Contact Us
  • Worldwide
Search
Click to view menu
MENUMENU
  • Products
    • Chromatography
      • HPLC
      • RHPLC
      • UHPLC
      • LC-MS
      • Preparative LC
      • Analytical SFC
      • Semi-Preparative SFC
      • Hybrid SFC
      • Fuel Analysis by SFC-FID
      • Preparative SFC
      • Supercritical Fluid Extraction
      • Chromatography Software
    • Molecular Spectroscopy
      • Circular Dichroism
      • High-Throughput CD
      • Vibrational CD
      • Circularly Polarized Luminescence (CPL)
      • Polarimeters
      • FTIR Spectrometers
      • FTIR Microscopy
      • FTIR Portable
      • Raman Microscopy
      • Palmtop Raman Spectrometer
      • Probe Raman
      • UV-Visible/NIR Spectrophotometers
      • UV-Visible/NIR Microscopy
      • Fluorescence Spectrophotometers
      • Film Thickness
      • Spectra Manager™ Suite
    • Refurbished
      • Refurbished HPLC Systems
      • HPLC Switching Valves
      • FTIR Accessories
  • Service
    • Service and Support Plans
    • Service Request Form
  • Applications
  • KnowledgeBase
  • Learning Center
    • Best Practice
      • Circular Dichroism Tips & Tricks for Biological Samples
      • CD Scale Calibration with ACS
      • Fluorescence Tips & Tricks
      • Raman Spectroscopy Tips & Tricks
    • Training Videos
      • ChromNAV
      • SF-NAV
      • Circular Dichroism
      • UV-Visible/NIR
      • Fluorescence
    • Training Seminars
      • Training Registration Form
    • Webinars
    • eBooks
    • Theory
      • Theory of Molecular Spectroscopy
      • Chromatography
  • About Us
    • President’s Message
    • Contact
    • History
    • Careers
  • News
  • Events
  • Worldwide
  • Shop
  • My Account
  • Contact Us

Home / Applications / Change in the Fluorescence Anisotropy Spectrum by the Denaturation of α-lactalbumin

  • Industry

  • Technique

Change in the Fluorescence Anisotropy Spectrum by the Denaturation of α-lactalbumin

By Heather Haffner

PDF IconDownload This Application

January 5, 2024

Introduction

Circular Dichroism spectroscopy is one of the leading techniques in protein structure analysis, with fluorescence spectroscopy and fluorescence anisotropy both providing complementary information. While CD spectra provide information regarding the secondary structure of proteins, fluorescence spectra and anisotropy provide information about the local environment surrounding the fluorophores. In particular, fluorescence anisotropy provides additional information about the rotational movement of these fluorophores which cannot be obtained by fluorescence spectroscopy alone.

The JASCO J-1500 CD spectrometer can be used to measure spectra for CD, absorption, fluorescence excitation and emission, and fluorescence anisotropy. This variety of techniques not only allows for secondary structure estimation but also the analysis of protein-ligand binding and rotational movement in proteins.

This application note describes the changes in fluorescence anisotropy measurements during the denaturation of α-lactalbumin by guanadinium hydrochloride (GuHCl).

J-1500 Circular Dichroism Spectrophotometer
J-1500 CD Spectrometer

Experimental

Measurement conditions
 Data acquisition interval 0.1 nm
 Excitation bandwidth 7 nm
 Response time 4 times
 Response time 2 sec
 Scan speed 100 nm

Keywords

210-CD-0032, J-1500, circular dichroism, CDF-426, fluorescence, anisotropy, α-lactalbumin, GuHCl, secondary structure, denaturation, biochemistry

Results

The fluorescence anisotropy spectra of both the native-state α-lactalbumin in H2O and the unfolded α-lactalbumin in 3.4 M GuHCl are shown in Figure 1. Both spectra show a peak maxima at 267 nm and peak minima at 283 and 291 nm. All three peaks result from the tryptophan residue of the protein. Figure 1 illustrates that the denaturation of α-lactalbumin clearly decreases the fluorescence anisotropy, indicating a more freely rotating tryptophan residue.

Fluorescence anisotropy spectra of the denaturation of α-lactalbumin by GuHCl. The green line indicates 0.02 mg/mL α-lactalbumin, 0.1 mM EDTA in the absence of GuHCl, and the blue line is 0.02 mg/mL α-lactalbumin, 0.1 mM EDTA in 3.4 M GuHCl.
Figure 1. Fluorescence anisotropy spectra of the denaturation of α-lactalbumin by GuHCl. The green line indicates 0.02 mg/mL α-lactalbumin, 0.1 mM EDTA in the absence of GuHCl, and the blue line is 0.02 mg/mL α-lactalbumin, 0.1 mM EDTA in 3.4 M GuHCl.

Conclusion

This application note demonstrates that the J-1500 CD spectrometer can be used to measure samples for a variety of spectroscopic techniques. The addition of the FPA-580 polarizer to the J-1500 spectrometer allows users to obtain CD and fluorescence anisotropy data all on the same instrument.

References

1. Canet, D., Doering, K., Dobson, C. M., and Y. Dupont, Biophysical Journal (2001), 80, 1996-2003.
2. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, New York, 446-487.

This document has been prepared based on information available at the time of publication and is subject to revision without notice. Although the contents are checked with the utmost care, we do not guarantee their accuracy or completeness. JASCO Corporation assumes no responsibility or liability for any loss or damage incurred as a result of the use of any information contained in this document. Copyright and other intellectual property rights in this document remain the property of JASCO Corporation. Please do not attempt to copy, modify, redistribute, or sell etc. in whole or in part without prior written permission.

Featured Products:

  • The basic, compact model for research, QA/QC, and teaching applications.

    J-1100

  • Highest performance with a wide range of accessories for maximum flexibility to meet complex research demands.

    J-1500

  • UV/Visible/NIR measurements up to 2,500 nm for MCD and specialized applications.

    J-1700

About the Author

JASCO Application Note

Change in the Fluorescence Anisotropy Spectrum by the Denaturation of α-lactalbumin

Introduction

Circular Dichroism spectroscopy is one of the leading techniques in protein structure analysis, with fluorescence spectroscopy and fluorescence anisotropy both providing complementary information. While CD spectra provide information regarding the secondary structure of proteins, fluorescence spectra and anisotropy provide information about the local environment surrounding the fluorophores. In particular, fluorescence anisotropy provides additional information about the rotational movement of these fluorophores which cannot be obtained by fluorescence spectroscopy alone.

The JASCO J-1500 CD spectrometer can be used to measure spectra for CD, absorption, fluorescence excitation and emission, and fluorescence anisotropy. This variety of techniques not only allows for secondary structure estimation but also the analysis of protein-ligand binding and rotational movement in proteins.

This application note describes the changes in fluorescence anisotropy measurements during the denaturation of α-lactalbumin by guanadinium hydrochloride (GuHCl).

J-1500 Circular Dichroism Spectrophotometer
J-1500 CD Spectrometer

Experimental

Measurement conditions
 Data acquisition interval 0.1 nm
 Excitation bandwidth 7 nm
 Response time 4 times
 Response time 2 sec
 Scan speed 100 nm

Results

The fluorescence anisotropy spectra of both the native-state α-lactalbumin in H2O and the unfolded α-lactalbumin in 3.4 M GuHCl are shown in Figure 1. Both spectra show a peak maxima at 267 nm and peak minima at 283 and 291 nm. All three peaks result from the tryptophan residue of the protein. Figure 1 illustrates that the denaturation of α-lactalbumin clearly decreases the fluorescence anisotropy, indicating a more freely rotating tryptophan residue.

Fluorescence anisotropy spectra of the denaturation of α-lactalbumin by GuHCl. The green line indicates 0.02 mg/mL α-lactalbumin, 0.1 mM EDTA in the absence of GuHCl, and the blue line is 0.02 mg/mL α-lactalbumin, 0.1 mM EDTA in 3.4 M GuHCl.
Figure 1. Fluorescence anisotropy spectra of the denaturation of α-lactalbumin by GuHCl. The green line indicates 0.02 mg/mL α-lactalbumin, 0.1 mM EDTA in the absence of GuHCl, and the blue line is 0.02 mg/mL α-lactalbumin, 0.1 mM EDTA in 3.4 M GuHCl.

Conclusion

This application note demonstrates that the J-1500 CD spectrometer can be used to measure samples for a variety of spectroscopic techniques. The addition of the FPA-580 polarizer to the J-1500 spectrometer allows users to obtain CD and fluorescence anisotropy data all on the same instrument.

Keywords

210-CD-0032, J-1500, circular dichroism, CDF-426, fluorescence, anisotropy, α-lactalbumin, GuHCl, secondary structure, denaturation, biochemistry

References

1. Canet, D., Doering, K., Dobson, C. M., and Y. Dupont, Biophysical Journal (2001), 80, 1996-2003.
2. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, New York, 446-487.

This document has been prepared based on information available at the time of publication and is subject to revision without notice. Although the contents are checked with the utmost care, we do not guarantee their accuracy or completeness. JASCO Corporation assumes no responsibility or liability for any loss or damage incurred as a result of the use of any information contained in this document. Copyright and other intellectual property rights in this document remain the property of JASCO Corporation. Please do not attempt to copy, modify, redistribute, or sell etc. in whole or in part without prior written permission.
28600 Mary’s Court, Easton, MD 21601 USA • (800) 333-5272 • Fax: (410) 822-7526 • jascoinc.com/applications

Close

Designed in Tokyo. TRUSTED globally.

View our support plans

Connect with JASCO

  • Facebook
  • Twitter
  • LinkedIn
  • JASCO Sales
  • 800-333-5272

Receive the latest promotions and special offers

  • This field is for validation purposes and should be left unchanged.
  • Careers
  • Press Kit
  • JASCO Privacy Policy
  • Sitemap
  • Environmental Policy

© , JASCO. All Rights Reserved.