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
||75W Xe Lamp (75W HgXe Lamp available for kinetics)
||180 - 900 nm using MgF2 optics
||5 - 95°C
||Millisecond deadtime (measure rate constants up to 1500 s-1)
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:
Protein Secondary Structure (260 nm to 180 nm) – Sample Preparation and Requirements
- 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)
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:
Protein Tertiary Structure (350 nm to 250 nm) – Sample Preparation and Requirements
- 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)
- Sample Purity
- Free of impurities
- Filtered (0.2 μm) and degassed prior to experiment
- Accurate protein concentrations
- Molar ellipticity conversions and secondary structure analysis software
- Quantitative amino acid composition
- Determination of backbone amide groups using the micro-biuret method.
- Determination of moles of tyrosine using difference spectroscopy under denaturing conditions.
- Determination of total nitrogen.
- Bradford Method
- Lowry Method
- Absorbance at 280 and/or 260 nm
- Choice of cuvette pathlength
- Quartz, short pathlength (0.01 cm)
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.