Circular dichroism
Introduction
Circular dichroism (CD) is a spectroscopic technique used to measure the difference in the absorption of left-handed and right-handed circularly polarized light by chiral molecules. This phenomenon is particularly useful in the study of optically active substances, including proteins, nucleic acids, and other biomolecules. CD spectroscopy provides insights into the secondary structure, folding, and conformational changes of these molecules, making it an invaluable tool in biochemistry and structural biology.
Principles of Circular Dichroism
Circular dichroism arises from the interaction of circularly polarized light with chiral molecules. When circularly polarized light passes through a chiral medium, the differential absorption of the two polarizations results in a measurable difference in intensity. This difference is quantified as ellipticity, which is directly related to the molecular structure of the sample.
Chiral Molecules and Optical Activity
Chirality is a geometric property of a molecule that makes it non-superimposable on its mirror image. Chiral molecules exhibit optical activity, meaning they can rotate the plane of polarized light. This property is fundamental to CD spectroscopy, as it allows for the differentiation between enantiomers, which are molecules that are mirror images of each other.
Circularly Polarized Light
Circularly polarized light consists of two perpendicular electromagnetic waves of equal amplitude but with a phase difference of 90 degrees. This results in a helical wavefront, which can be either left-handed or right-handed. In CD spectroscopy, the differential absorption of these two types of circularly polarized light by a chiral sample is measured.
Instrumentation and Measurement
CD spectrometers are designed to measure the ellipticity of a sample over a range of wavelengths. The key components of a CD spectrometer include a light source, monochromator, polarizer, sample holder, and detector.
Light Source
The light source in a CD spectrometer typically emits ultraviolet (UV) or visible light, depending on the sample being studied. Deuterium and xenon lamps are commonly used due to their broad emission spectra and stability.
Monochromator
A monochromator is used to select specific wavelengths of light from the source. This is crucial for obtaining accurate CD spectra, as different molecular transitions occur at different wavelengths.
Polarizer
The polarizer converts linearly polarized light into circularly polarized light. This is achieved using a photoelastic modulator or a Pockels cell, which can rapidly switch between left-handed and right-handed circular polarization.
Sample Holder
The sample holder is typically a quartz cuvette, which is transparent to UV and visible light. The path length of the cuvette can vary, but it is usually between 0.1 mm and 10 mm, depending on the concentration and absorption characteristics of the sample.
Detector
The detector measures the intensity of light passing through the sample. Photomultiplier tubes or photodiodes are commonly used due to their sensitivity and fast response times.
Applications of Circular Dichroism
CD spectroscopy is widely used in the analysis of biomolecules, providing valuable information about their structure and dynamics.
Protein Secondary Structure
One of the primary applications of CD is the determination of protein secondary structure. By analyzing the CD spectrum in the far-UV region (190-250 nm), researchers can estimate the content of alpha-helices, beta-sheets, and random coils in a protein. This information is crucial for understanding protein folding and function.
Nucleic Acids
CD spectroscopy is also used to study nucleic acids, such as DNA and RNA. The CD spectra of these molecules provide insights into their helical structure, base stacking, and conformational changes. This is particularly important in the study of DNA-protein interactions and the effects of drugs on nucleic acid structure.
Conformational Changes and Folding
CD can monitor conformational changes in biomolecules, such as protein folding and unfolding. By measuring CD spectra at different temperatures or in the presence of denaturants, researchers can study the stability and folding pathways of proteins.
Chirality and Enantiomeric Purity
CD is a powerful tool for determining the chirality and enantiomeric purity of compounds. This is especially important in the pharmaceutical industry, where the biological activity of a drug can depend on its chirality.
Advantages and Limitations
CD spectroscopy offers several advantages, but it also has limitations that must be considered.
Advantages
- **Non-destructive:** CD is a non-destructive technique, allowing for the analysis of precious or limited samples. - **Rapid and Sensitive:** CD measurements are quick and can detect subtle changes in molecular structure. - **Applicable to a Wide Range of Samples:** CD can be used to study a variety of chiral molecules, including proteins, nucleic acids, and small organic compounds.
Limitations
- **Limited Structural Resolution:** CD provides information about overall secondary structure but lacks the detailed resolution of techniques like X-ray crystallography or NMR spectroscopy. - **Sample Requirements:** CD requires optically transparent samples, which can limit its applicability to highly absorbing or turbid samples. - **Interpretation Complexity:** The interpretation of CD spectra can be complex, requiring careful analysis and comparison with known standards.