Circular Dichroism

Circular dichroism (CD) spectroscopy measures differences in the absorption of left-handed versus right-handed polarized light that arise due to structural asymmetry. CD spectroscopy has become a very useful technique for characterization of biomolecules.

The PiStar-180 spectrometer available at the SSSC is designed for both steady-state and kinetic CD applications. In addition to CD detection, the PiStar-180 spectrometer can also do measurements in absorbance or fluorescence modes.

Applied Photophysics Pistar-180 Circular Dichroism

Circular dichroism is a spectroscopic technique that measures the difference in the absorption of left-handed and right-handed circularly polarized light. The difference in the absorption between left and right circularly polarized light is due to a molecule’s structural asymmetry. The Pistar-180 CD spectrometer (Applied Photophysics, Leatherhead, UK) can be used to make measurements in the far-UV to NIR spectral regions (180-1000 nm) at variable temperature. The Pistar 180 is also equipped for absorbance, total fluorescence and partial fluorescence, and stop-flow kinetics measurements.

Information obtained from CD measurements include:

  • Protein folding – evaluation of secondary structure, tertiary structure
  • Conformation stability – evaluation of proteins stability to thermal stability, pH stability, and stability to denaturants
  • Solution effects (buffers, salts, detergents, etc.) on the conformation of biomolecules
  • Kinetic information on protein folding, denaturation, etc.
  • Determination of protein-protein interactions and their effects on protein conformation

Specifications

Light Source 75W Xe Lamp (75W HgXe Lamp available for kinetics)
Wavelength Range 180 - 900 nm using MgF2 optics
Temperature Range 5 - 95°C
Kinetics Millisecond deadtime (measure rate constants up to 1500 s-1)

Settings

In general, samples should be prepared the same way as absorbance measurements (i.e. follow the Beer-Lambert Law - A = εcb). A general checklist for sample preparation should include:

  • Solvent should be transparent in spectral region of interest
  • Absorbance of interested CD active peaks should not exceed 1.2
  • Samples should be pure, filtered, and degassed before use
  • The choice of cuvette will depend upon spectral region of interest and solvent transparency (e.g. b = 0.01 cm used in far-UV; b = 1 cm used in near-UV)

Protein Secondary Structure (260 nm to 180 nm) – Sample Preparation and Requirements
Typically, a concentration of 1 mg mL-1 (b = 0.01 cm, V = 50 μL) of protein is a useful starting concentration when first starting CD measurements, although it will all depend on the absorbance of your sample in the far-UV (Abs < 1.2).

Other considerations include:

  1. Choice of solvent/buffer
    • Minimize ionic strength (10mM good for most cases)
    • Solvent transparency considerations
    • Phosphate buffers are preferable, AVOID TRIS
    • Avoid Cl- ions (substitute with F-, SO4-2 anions)
  2. Sample Purity
    • Free of impurities
    • Filtered (0.2 μm) and degassed prior to experiment
  3. Accurate protein concentrations
    • Molar ellipticity conversions and secondary structure analysis software

    Good Methods
    1. Quantitative amino acid composition
    2. Determination of backbone amide groups using the micro-biuret method.
    3. Determination of moles of tyrosine using difference spectroscopy under denaturing conditions.
    4. Determination of total nitrogen.
    Not Acceptable
    1. Bradford Method
    2. Lowry Method
    3. Absorbance at 280 and/or 260 nm
  4. Choice of cuvette pathlength
    • Quartz, short pathlength (0.01 cm)

Protein Tertiary Structure (350 nm to 250 nm) – Sample Preparation and Requirements
Typically, a concentration of 1 mg mL-1 (b = 1 cm, V = 1000 μL) of protein is a useful starting concentration when first starting CD measurements in the near-UV, although it will all depend on the absorbance of your sample (Abs < 1.2). The same considerations as that listed in the protein secondary structure should be followed, with the exception of the choice of solvent/buffer. Most solvent/buffers are transparent in this spectral region.

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