INTRODUCTION
Molecular analysis using circular dichroism spectroscopy and optical rotatory dispersion is a major helpful technique in biology. Circular Dichroism (CD) and Optical Rotatory Dispersion (ORD) are analytical techniques used to investigate the structural and functional characteristics of biological molecules such as proteins, nucleic acids, and lipids. These techniques are based on the differential absorption of left and right-handed circularly polarized light by chiral molecules.
CD measures the difference in the absorbance of left and right-handed circularly polarized light as it passes through a sample. It provides information about the secondary and tertiary structures of proteins and nucleic acids, such as the presence of alpha-helices, beta-sheets, and random coils. CD can also be used to study conformational changes in proteins induced by ligand binding or changes in environmental conditions.
ORD, on the other hand, measures the rotation of the plane of polarization of linearly polarized light as it passes through a chiral sample. It provides information about the primary structure of molecules, such as the sequence of amino acids in a protein.
CD and ORD are powerful tools for studying biomolecules because they are sensitive to even small structural changes, can be used in solution or in solid-state samples, and require minimal sample preparation. They are widely used in biochemistry, biophysics, and molecular biology research to study protein folding, ligand binding, enzyme kinetics, and molecular interactions.
PRINCIPLE AND WORKING
Circular Dichroism (CD) and Optical Rotatory Dispersion (ORD) are based on the fact that chiral molecules can interact differently with left and right-handed circularly polarized light due to their asymmetry. CD measures the difference in absorption of left and right-handed circularly polarized light by a sample, while ORD measures the rotation of the plane of polarization of linearly polarized light by a sample.
In CD spectroscopy, a sample is placed in a cuvette between two polarizers that transmit linearly polarized light. The incident light is then passed through a quarter-wave plate that converts it into circularly polarized light. As the light passes through the sample, it interacts with chiral molecules in the sample, resulting in differential absorption of left and right-handed circularly polarized light. The transmitted light is then detected by a detector, and the difference in absorbance between left and right-handed polarized light is measured. This difference in absorbance is proportional to the circular dichroism of the sample.
In ORD spectroscopy, a sample is also placed in a cuvette between two polarizers that transmit linearly polarized light. The incident light is then passed through the sample, and the rotation of the plane of polarization of the light passing through the sample is measured. The angle of rotation is proportional to the optical rotation of the sample.
CD and ORD spectroscopy are highly sensitive to the conformational changes in chiral molecules, making them powerful tools for studying biomolecules such as proteins, nucleic acids, and lipids. CD can be used to study the secondary and tertiary structures of proteins and nucleic acids, while ORD can be used to study the primary structure of molecules, such as the sequence of amino acids in a protein.
EXPRESSION AND DERIVATION
Circular dichroism and optical rotation of a chiral molecule can be expressed in terms of the molar ellipticity and specific rotation, respectively. The molar ellipticity (θ) is the difference in absorbance of left- and right-handed circularly polarized light per unit length and concentration of the chiral species, and is calculated using the equation θ = Δε / l c,
where Δε is the difference in molar absorption coefficients of left- and right-handed circularly polarized light.
CD is related to Δε and can be expressed as CD = (A_L – A_R) / A_0, where A_L and A_R are the absorbances of left- and right-handed circularly polarized light, and A_0 is the average absorbance of the two.
The molar ellipticity can be calculated from CD and the average molar absorption coefficient using the equation
θ = (CD * ε_0) / (l c).
These interactions are described by the Maxwell equations of electromagnetic theory and require specialized instruments such as CD and ORD spectrometers that can generate and detect polarized light.
INSTRUMENTATION OF CIRCULAR DICHROISM SPECTROSCOPY
Circular dichroism (CD) and optical rotation dispersion (ORD) spectrometers are specialized instruments used to measure the interaction of chiral molecules with polarized light. These instruments typically consist of a light source, monochromator, polarizer, sample holder, and detector.
The light source emits a broad range of wavelengths of light, which is then filtered to a narrow band of wavelengths by the monochromator. The polarizer then polarizes the light, ensuring that only circularly or linearly polarized light passes through the sample. The sample holder holds the sample in place and allows the polarized light to pass through it.
For CD spectrometers, the detector measures the intensity of light that passes through the sample, allowing for the measurement of the molar ellipticity of the sample. For ORD spectrometers, a second polarizer, called an analyzer, is added to measure the angle of rotation of the linearly polarized light passing through the sample.
Advanced optics, detectors, and electronics are required for these instruments to measure the differences in polarization of light caused by chiral molecules in a sample. These instruments are invaluable tools for researchers studying the properties and behaviors of chiral molecules in a variety of fields, including chemistry, biochemistry, and pharmaceuticals.
APPLICATION IN BIOLOGY
Circular dichroism (CD) and optical rotation dispersion (ORD) spectroscopy have a wide range of applications in the field of biology. These techniques are used to investigate the structures, conformations, and interactions of biomolecules, including proteins, nucleic acids, carbohydrates, and lipids.
Proteins are the most commonly studied biomolecules using CD and ORD spectroscopy. CD spectroscopy is used to investigate the secondary and tertiary structures of proteins. The spectra obtained from CD measurements can be used to determine the alpha-helical, beta-sheet, and random coil content of a protein. Additionally, CD spectroscopy can be used to study protein conformational changes induced by changes in pH, temperature, and ligand binding. ORD spectroscopy can be used to measure the absolute configuration of chiral amino acids in proteins.
- CD and ORD spectroscopy can also be used in the development of new biotech materials. For example, CD spectroscopy can be used to investigate the self-assembly of peptides into nanostructures. The spectra obtained from CD measurements can be used to determine the secondary structure of the peptides and to monitor the formation of the nanostructures. This information can be used to design new materials with specific properties, such as drug delivery systems or tissue engineering scaffolds.
- Another application of CD and ORD spectroscopy in biotechnology is the study of protein-protein interactions. CD spectroscopy can be used to investigate the binding of proteins to each other, and the spectra obtained can be used to determine the changes in protein conformation induced by binding. This information can be used to develop new biotech products, such as biosensors or diagnostic assays.
Nucleic acids, including DNA and RNA, can also be studied using CD spectroscopy. The spectra obtained from CD measurements of nucleic acids can be used to determine the secondary structure of these molecules, including the presence of single-stranded or double-stranded regions, and the presence of base-pairing interactions.
Carbohydrates, including polysaccharides and glycoproteins, can also be studied using CD spectroscopy. The spectra obtained from CD measurements can be used to determine the stereochemistry of carbohydrates, including the configuration of glycosidic bonds and the conformation of the molecule.
Lipids, including phospholipids and glycolipids, can also be studied using CD spectroscopy. The spectra obtained from CD measurements can be used to determine the chirality of lipid molecules and to investigate the effects of membrane composition on lipid structure.
CONCLUSION
In conclusion, circular dichroism (CD) and optical rotation dispersion (ORD) are important techniques used to study the structure and behavior of chiral molecules in biology and biotechnology. These techniques provide valuable information on the secondary structure and conformational changes of proteins, peptides, nucleic acids, and other chiral molecules, as well as their interactions with other molecules. The application of CD and ORD spectroscopy has led to advances in drug development, material science, and biotechnology, and will continue to be an important tool for researchers in these fields. With the continued development of instrumentation and techniques, CD and ORD spectroscopy will play an increasingly important role in understanding the complex biological and biochemical processes that underlie life and disease.