Introduction
Circular dichroism (CD) is form of light absorption spectroscopy that measures the difference in absorbance of right- and left-circularly polarized light (rather than the commonly used absorbance of isotropic light) by a substance. It is applicable for molecules have one or more chiral chromophores [1].
Circular dichroism = ”A(”) = A(”)LCPL – A(”)RCPL, where ” is the wavelength
This technique measured a molecule over a range of wavelengths. All chiral molecules can be studied, particularly in study of large biological molecules. A primary application is in the analysing the conformation of macromolecules or secondary structure (particularly proteins). Circular dichroism is used to measure the changes of secondary structure with environmental conditions or on interaction with other molecules. kinetic and thermodynamic macromolecules can be studied by CD spectroscopy.
The Basics of CD
A quarter-wave plate is term applied when the optical element converts between linearly polarised light and circularly polarised light. It is also known as birefringent (the refractive indexes seen by horizontally and vertically polarised light are different). Slowing one of the linear components of the beam, oriented plate will convert linearly polarised light into circularly polarised light. A beam (left- or right-CPL) will produced [1].
The basis of circular dichroism is the difference in the absorbance of left- and right-CPL. A molecule that absorbs LCP and RCP differently is considered as optically active or chiral molecule.
At light wavelengths that can be absorbed by a chiral molecule, Circular dichroism is occur. Absorption may occur at different extents (e.g., 90% of R-CPL and 88% of L-CPL may absorbed by chiral chromophore). The primary spectroscopic property measured by CD spectrometer is the Chirascan. Thus, Circular dichroism measured as a function of wavelength.
Optical rotation (ORD) or circular dichroism (CD) can be calculated from the other if spectral information of ORD or CD is available. CD spectra is better resolved spectrally than optical rotation spectra.
Circular birefringence and optical rotation
Presence of anisotropic medium of chiral substance with L-CPL and R-CPL that propagate at different speeds will lead to exhibit circular birefringence by chiral molecules
Circular Dichroism and the Study of Biological Molecules
The phenomena of Interaction of polarised light with chiral molecules is basis of Circular dichroism. Highly chiral molecules in the biological molecules make it the main example for application of CD (19 of 20 amino acids that form proteins have a chiral molecule)
Structures of chiral macromolecules such as proteins and DNA can be studied by circular dichroism. CD spectrum of macromolecules is not a sum of the CD spectra of the individual residues or bases. Structure of 3-dimension of the macromolecule is greatly influenced and specific circular dichroism signature for each structure is appear.
CD is excellent tool to monitor dynamic changes in structure of protein. The changes can be induced by changing in temperature, pH, ligands, or denaturants.
Determination of Protein Secondary Structure by Circular Dichroism:
CD spectroscopy at far-UV region (190-250nm) is used to detect protein of Secondary structure. Peptide bond is the chromophore At this region. Alpha-helix, beta-sheet, and random coil structures make the CD spectrum characteristic shape and magnitude. About 20 up to 200 ”l of solution containing 1 mg/ml to 50 ”g/ml protein is required.
Information About Protein Tertiary Structure from Circular Dichroism:
CD spectroscopy at near-UV region (250 nm – 350 nm) is used to detect protein of tertiary structure. Aromatic amino acids and disulphide bonds are the chromophores at this region. The spectrum is sensitive to variations in tertiary structure due to interactions and variations in conditions of the solvent.
Signals in (250 nm-270 nm) are applicable for phenylalanine residues, (270 nm-290 nm) are applicable for tyrosine, and (280 nm-300 nm) are applicable for tryptophan. Around 1 ml of protein solution is required (0.25 to 2 mg/ml for most proteins).
Demonstrating Comparability of Conformation:
It is important to study the different lots of a protein to ensure they have the equivalent conformations.
Thermal Stability by Circular Dichroism
Studying the thermal stability that affected by variations such as temperature, pH, buffers, sugars, amino acids, or salts can be assessed by CD.
Melting of Tertiary Structure
Measuring variation in near-UV region (tertiary structure changes) lead to higher concertation of protein (1-3 mg) is required due to the weakness in signal in this region. Single or two step reaction in such studies is occurred for melting of a protein.
Melting of Protein Complexes
The effect of forming a protein-protein on the thermal stability of the individual proteins in the complex can also be determined.
Nucleic acids and other biomolecules
Any chiral biomolecule or biomolecule can be analyzed and yield similar data to those explained above for peptides and proteins. For example, CD spectroscopy was used to identify the rare Z-form of DNA and to measure the binding of RNA aptamers to their targets.
CD Spectrometer Performance
Signal-to-noise (S/N) is the characteristic that used to determine the limit of detection of CD spectrophotometer. Better the S/N is means better in limit of detection.
A measurement of long period of time must be considered to determine the true average. The time is inversely proportional to S/N. thus, maximum S/N required for designing optimum circular dichroism spectrophotometer.
S/N can be enhanced by increasing the light intensity of incident linearly-polarised, increasing the efficiency of the detector, or averaging and collecting data points in long time.
Sample preparation and measurement
A buffer or detergent or other chemical should not be used unless it will not mask the signal of protein. In addition, compound that could be absorbed in the desired region (190 – 250 nm) should not be used. However, Protein solution should contain only chemicals (with its lowest concentrations) that maintain protein stability. Pure protein should be used if possible, hence any additional peptide or protein will interfere with the CD signal. Noise of signal will appear in the presence of Unfolded protein, peptides, scattering particles . Filtering of the solutions by 0.02 um filters may improve signal to noise ratio.