Circular dichroism (CD) is a form of spectroscopy based on the differential absorption of left- and right-handed circularly polarized light. It can be used to determine the secondary structure of proteins. Extremely high resolution spectrogram of the Sun showing thousands of elemental absorption lines (fraunhofer lines) Spectroscopy is the study of the interaction between radiation (electromagnetic radiation, or light, as well as particle radiation) and matter. ... In electrodynamics, circular polarization of electromagnetic radiation is a polarization such that the tip of the electric field vector, at a fixed point in space, describes a circle as time progresses. ... This article does not cite any references or sources. ... A representation of the 3D structure of the Myoglobin protein. ... A representation of the 3D structure of myoglobin, showing coloured alpha helices. ...

Polarized Light

Linearly polarized light is polarized in a certain direction (i.e. the magnitude of its electric field vector oscillates only in one plane, similar to a sine wave). In circularly polarized light the electric field vector has a constant length, but rotates about its propagation direction. Hence, it forms a helix in space while propagating. If this is a left-handed helix the light is referred to as left circularly polarized and vice versa for a right handed helix. See external links for a demonstrative animation of the different types of electromagnetic waves.

The electric field of a light beam causes a linear displacement of charge when interacting with a molecule, whereas the magnetic field of it causes a circulation of charge. These two motions combined result in a helical displacement when light is impinged on a molecule. Since circularly polarized light itself is "chiral", it interacts differently with chiral molecules. That is, one of the two types of circularly polarized light are absorbed to different extents. In a CD experiment, equal amounts of left and right circularly polarized light are radiated into a (chiral) solution. One of the two types is absorbed more than the other one and this wavelength dependent difference of absorption is measured, yielding the CD spectrum of the sample.

Due to the interaction with the molecule, the electric field vector of the light traces out an elliptical path while propagating.

Derivation of ellipticity

At a given wavelength, The wavelength is the distance between repeating units of a wave pattern. ...

where ΔA is the difference between absorbance of left circularly polarized (LCP) and right circularly polarized (RCP) light (this is what is usually measured).

It can also be expressed, by applying Beer's law, as: In optics, the Beer-Lambert law, also known as Beers law or the Lambert-Beer law or the Beer-Lambert-Bouguer law is an empirical relationship that relates the absorption of light to the properties of the material through which the light is traveling. ...

where

ε_L and ε_R are the molar extinction coefficients for RCP and LCP light,

C is the molar concentration

l is the path length in centimeters (cm).

Then

is the molar circular dichroism. This is what is usually meant by the circular dichroism of the substance. Although ΔA is usually measured, for historical reasons most measurements are reported in degrees of ellipticity. Molar circular dichroism and molar ellipticity, [θ], are readily interconverted by the equation:

Elliptical polarized light (purple) is composed of unequal contributions of right (blue) and left (red) circular polarized light.

.

This relationship is derived by defining the ellipticity of the polarization as: Image File history File links Electric_Vectors_1. ... Image File history File links Electric_Vectors_1. ... In electrodynamics, polarization (also spelled polarisation) is the property of electromagnetic waves, such as light, that describes the direction of their transverse electric field. ...

where

E_R and E_L are the magnitudes of the electric field vectors of the right-circularly and left-circularly polarized light, respectively.

When E_R equals E_L (when there is no difference in the absorbance of right- and left-circular polarized light), θ is 0° and the light is linearly polarized. When either E_R or E_L is equal to zero (when there is complete absorbance of the circular polarized light in one direction), θ is 45° and the light is circularly polarized. In physics, the space surrounding an electric charge or in the presence of a time-varying magnetic field has a property called an electric field. ... A vector going from A to B. In physics and in vector calculus, a spatial vector, or simply vector, is a concept characterized by a magnitude and a direction. ... In electrodynamics, linear polarization or plane polarization of electromagnetic radiation is a confinement of the electric field vector or magnetic field vector to a given plane along the direction of propagation. ... In electrodynamics, circular polarization of electromagnetic radiation is a polarization such that the tip of the electric field vector, at a fixed point in space, describes a circle as time progresses. ...

Generally, the circular dichroism effect is small, so tanθ is small and can be approximated as θ in radians. Since the intensity or irradiance, I, of light is proportional to the square of the electric-field vector, the ellipticity becomes: In mathematics and physics, the radian is a unit of angle measure. ... In physics, intensity is a measure of the time-averaged energy flux. ... Irradiance, radiant emittance, and radiant exitance are radiometry terms for the power of electromagnetic radiation at a surface, per unit area. ...

Then by substituting for I using Beer's Law in natural logarithm form: In optics, the Beer-Lambert law, also known as Beers law or the Lambert-Beer law or the Beer-Lambert-Bouguer law is an empirical relationship that relates the absorption of light to the properties of the material through which the light is traveling. ... The natural logarithm, formerly known as the hyperbolic logarithm, is the logarithm to the base e, where e is equal to 2. ...

The ellipticity can now be written as:

Since ΔA<<1, this expression can be approximated by expanding the exponentials in a Taylor series to first-order and then discarding terms of ΔA in comparison with unity and converting from radians to degrees: As the degree of the Taylor series rises, it approaches the correct function. ... In mathematics and physics, the radian is a unit of angle measure. ...

The linear dependence of solute concentration and pathlength is removed by defining molar ellipticity as,

Then combining the last two expression with Beer's Law, molar ellipticity becomes: In optics, the Beer-Lambert law, also known as Beers law or the Lambert-Beer law or the Beer-Lambert-Bouguer law is an empirical relationship that relates the absorption of light to the properties of the material through which the light is traveling. ...

Application to biological molecules

In general, this phenomenon will be exhibited in absorption bands of any optically active molecule. As a consequence, circular dichroism is exhibited by biological molecules, because of the dextrorotary (e.g. some sugars) and levorotary (e.g. some amino acids) molecules they contain. Even more important is that a secondary structure will also impart a distinct CD to its respective molecules. Therefore, the alpha helix of proteins and the double helix of nucleic acids have CD spectral signatures representative of their structures. When polarized light is passed through a substance containing chiral molecules (or nonchiral molecules arranged asymmetrically), the direction of polarization can be changed. ... In chemistry, two stereoisomers are said to be enantiomers if they are mirror images of each other. ... Magnification of grains of sugar, showing their monoclinic hemihedral crystalline structure. ... In chemistry, two stereoisomers are said to be enantiomers if they are mirror images of each other. ... Phenylalanine is one of the standard amino acids. ... A representation of the 3D structure of the Myoglobin protein. ... Side view of an α-helix of alanine residues in atomic detail. ... The Double-Helix are an alien race in the Wing Commander science fiction series. ... Highly simplified diagram of a double-stranded nucleic acid. ...

CD is closely related to the optical rotatory dispersion (ORD) technique, and is generally considered to be more advanced. CD is measured in or near the absorption bands of the molecule of interest, while ORD can be measured far from these bands. In principle these two spectral measurements can be interconverted through an integral transform, if all the absorptions are included in the measurements. Optical rotatory dispersion is the variation in the optical rotation of a substance with a change in the wavelength of light. ...

The far-UV (ultraviolet) CD spectrum of proteins can reveal important characteristics of their secondary structure. CD spectra can be readily used to estimate the fraction of a molecule that is in the alpha-helix conformation, the beta-sheet conformation, the beta-turn conformation, or some other (e.g. random coil) conformation. These fractional assignments place important constraints on the possible secondary conformations that the protein can be in. CD cannot, in general, say where the alpha helices that are detected are located within the molecule or even completely predict how many there are. Despite this, CD is a valuable tool, especially for showing changes in conformation. It can, for instance, be used to study how the secondary structure of a molecule changes as a function of temperature or of the concentration of denaturing agents, e.g. Guanidinium hydrochloride or urea. In this way it can reveal important thermodynamic information (such as the enthalpy and Gibbs free energy of denaturation) about the molecule that cannot otherwise be easily obtained. Anyone attempting to study a protein will find CD a valuable tool for verifying that the protein is in its native conformation before undertaking extensive and/or expensive experiments with it. Also, there are a number of other uses for CD spectroscopy in protein chemistry not related to alpha-helix fraction estimation. “UV” redirects here. ... A representation of the 3D structure of the Myoglobin protein. ... Side view of an α-helix of alanine residues in atomic detail. ... Diagram of β-pleated sheet with H-bonding between protein strands The β sheet (also β-pleated sheet) is the second form of regular secondary structure in proteins — the first is the alpha helix — consisting of beta strands connected laterally by three or more hydrogen bonds, forming a generally twisted, pleated sheet. ... A turn is an element of secondary structure in proteins. ... Illustration of a 3-dimensional polypeptide A random coil is a polymer conformation where the monomer subunits are oriented randomly while still being bonded to adjacent units. ... Guanidine is a crystalline compound of strong alkalinity formed by the oxidation of guanine. ... Urea is an organic compound with the chemical formula (NH2)2CO. Urea is also known as carbamide, especially in the recommended International Nonproprietary Names (rINN) in use in Europe. ... t In thermodynamics and molecular chemistry, the enthalpy or heat content (denoted as H or ΔH, or rarely as χ) is a quotient or description of thermodynamic potential of a system, which can be used to calculate the useful work obtainable from a closed thermodynamic system under constant pressure. ... In thermodynamics, the Gibbs free energy is a thermodynamic potential which measures the useful work obtainable from a closed thermodynamic system at a constant temperature and pressure. ...

The near-UV CD spectrum (>250 nm) of proteins provides information on the tertiary structure. The signals obtained in the 250-300 nm region are due to the absorption, dipole orientation and the nature of the surrounding environment of the phenylalanine, tyrosine, cysteine (or S-S disulphide bridges) and tryptophan amino acids. Unlike in far-UV CD, the near-UV CD spectrum cannot be assigned to any particular 3D structure. Near-UV CD spectra can also provide structural information on the nature of the prosthetic groups in proteins, such as heme groups e.g. in hemoglobin and cytochrome c.

CD gives less specific structural information than e.g. X-ray crystallography or protein NMR spectroscopy, that both give atomic resolution data. However CD spectroscopy is a quick method, that does not require large amounts of proteins and extensive data processing. Thus CD can be used to survey a large number of solvent conditions, varying temperature, pH, salinity and presence of various cofactors. X-ray crystallography, also known as single-crystal X-ray diffraction, is the oldest and most common crystallographic method for determining the structure of molecules. ... Pacific Northwest National Laboratorys high magnetic field (800 MHz) NMR spectrometer being loaded with a sample. ... A solvent is a liquid that dissolves a solid, liquid, or gaseous solute, resulting in a solution. ... This article includes a list of works cited or a list of external links, but its sources remain unclear because it lacks in-text citations. ... The correct title of this article is . ... Annual mean sea surface salinity for the World Ocean. ...

CD spectroscopy is usually used to study proteins in solution, and thus it complements methods that study the solid state. This is also a limitation, in that many proteins are embedded in membranes in their native state, and solutions containing membrane structures are often strongly scattering. CD is sometimes measured in thin films. Extremely high resolution spectrogram of the Sun showing thousands of elemental absorption lines (fraunhofer lines) Spectroscopy is the study of the interaction between radiation (electromagnetic radiation, or light, as well as particle radiation) and matter. ... A biological membrane or biomembrane is an enclosing or separating tissue which acts as a barrier within or around a cell. ...

Experimental limitations

CD has also been studied in carbohydrates, but with limited success due to the experimental difficulties associated with measurement of CD spectra in the vacuum ultraviolet (VUV) region of the spectrum (100-200 nm), where the corresponding CD bands of unsubstituted carbohydrates lie. Substituted carbohydrates with bands above the VUV region have been successfully measured. Carbohydrates (literally hydrates of carbon) are chemical compounds that act as the primary biological means of storing or consuming energy, other forms being fat and protein. ...

Measurement of CD is also complicated by the fact that typical aqueous buffer systems often absorb in the range where structural features exhibit differential absorption of circularly polarized light. Phosphate, sulfate, carbonate, and acetate buffers are generally incompatible with CD unless made extremely dilute e.g. in the 10-50 mM range. The TRIS buffer system should be completely avoided when performing far-UV CD. Borate and ammonium salts are often used to establish the appropriate pH range for CD experiments. Some experimenters have substituted fluoride for chloride ion because fluoride absorbs less in the far UV, and some have worked in pure water. Another, almost universal, technique is to minimize solvent absorption by using shorter path length cells when working in the far UV, 0.1 mm path lengths are not uncommon in this work. Above is a ball-and-stick model of the inorganic hydrogenphosphate anion (HPO42−). Colour coding: P (orange); O (red); H (white). ... In inorganic chemistry, a sulfate (IUPAC-recommended spelling; also sulphate in British English) is a salt of sulfuric acid. ... In organic chemistry, a carbonate is a salt of carbonic acid. ... An acetate, or ethanoate, is a salt or ester of acetic acid. ... Borates in chemistry are chemical compounds containing boron bonded to three oxygen atoms written as B(OR)3. ...

In addition to measuring in aqueous systems, CD, particularly far-UV CD, can be measured in organic solvents e.g. ethanol, methanol,trifluoroethanol (or TFE). The latter has the advantage to induce structure formation of proteins, inducing beta-sheets in some and alpha helices in others, which they would not show under normal aqueous conditions. Most common organic solvents such as acetonitrile, THF, chloroform, dichloromethane are however, incompatible with far-UV CD. 2,2,2-Trifluoroethanol (TFE) or trifluoroethyl alcohol is a trifluoro substituted alcohol. ... Possible meanings: Task Force Europe Tetrafluoroethylene Thin Film Equipment The Fifth Element The Final Experiment Category: ... Acetonitrile is an organic molecule, often used as a solvent, with the chemical formula of CH3CN. Also known as methyl cyanide, it is the simplest of the organic nitriles. ... Tetrahydrofuran, also known as THF, hydrofuran, oxolane, oxacyclopentane, or furanidine, is a heterocyclic organic compound. ... R-phrases , , , S-phrases , Flash point Non-flammable U.S. Permissible exposure limit (PEL) 50 ppm (240 mg/m3) (OSHA) Supplementary data page Structure and properties n, εr, etc. ... Dichloromethane or Methylene chloride is a chemical compound widely used as a solvent for organic materials. ...

It may be of interest to note that the protein CD spectra used in secondary structure estimation are related to the π to π* orbital absorptions of the amide bonds linking the amino acids. These absorption bands lie partly in the so-called vacuum ultraviolet (wavelengths less than about 200 nm). The wavelength region of interest is actually inaccessible in air because of the strong absorption of light by oxygen at these wavelengths. In practice these spectra are measured not in vacuum but in an oxygen-free instrument (filled with pure nitrogen gas). General Name, Symbol, Number oxygen, O, 8 Chemical series nonmetals, chalcogens Group, Period, Block 16, 2, p Appearance colorless (gas) very pale blue (liquid) Standard atomic weight 15. ... General Name, Symbol, Number nitrogen, N, 7 Chemical series nonmetals Group, Period, Block 15, 2, p Appearance colorless gas Standard atomic weight 14. ...

Once oxygen has been eliminated, perhaps the second most important technical factor in working below 200 nm is to design the rest of the optical system to have low losses in this region. Critical in this regard is the use of aluminized mirrors whose coatings have been optimized for low loss in this region of the spectrum. A mirror is a reflective surface that is smooth enough to form an image. ...

The usual light source in these instruments is a high pressure, short-arc xenon lamp. Ordinary xenon arc lamps are unsuitable for use in the low UV. Instead specially constructed lamps with envelopes made from high-purity synthetic fused silica must be used. Light from synchrotron sources has a much higher flux at short wavelengths, and has been used to record CD down to 160 nm. General Name, Symbol, Number xenon, Xe, 54 Chemical series noble gases Group, Period, Block 18, 5, p Appearance colorless Standard atomic weight 131. ... Fused quartz is a man-made material manufactured principally from sands. ... Synchrotrons are now mostly used for producing monochromatic high intensity X-ray beams; here, the synchrotron is the circular track, off which the beamlines branch. ...

At the quantum mechanical level, the information content of circular dichroism and optical rotation are identical. Fig. ... When polarized light is passed through a substance containing chiral molecules (or nonchiral molecules arranged asymmetrically), the direction of polarization can be changed. ...

See also

• Dichroism
• Magnetic circular dichroism
• Optical activity
• Optical isomerism
• Optical rotation
• Polarization

In optics, the term dichroic has two related but distinct meanings. ... Magnetically induced circular dichroism (MCD), is the differential absorption of left and right circularly polarized light in the presence of a magnetic field. ... When polarized light is passed through a substance containing chiral molecules (or nonchiral molecules arranged asymmetrically), the direction of polarization can be changed. ... Optical isomerism is a form of isomerism (specifically stereoisomerism) where the two different isomers are the same in every way except being non-superposable mirror images of each other. ... When polarized light is passed through a substance containing chiral molecules (or nonchiral molecules arranged asymmetrically), the direction of polarization can be changed. ... In electrodynamics, polarization (also spelled polarisation) is the property of electromagnetic waves, such as light, that describes the direction of their transverse electric field. ...

External links

• Circular Dichroism explained - A commercial supplier's site, but very good information with examples, etc.
• Circular Dichroism at UMDNJ - a good site for information on structure estimation software
• Electromagnetic waves - Animated electromagnetic waves

References

1. Fasman, G.D., Circular Dichroism and the Conformational Analysis of Biomolecules (1996) Plenum Press, New York.
2. Hecht, E., Optics 3^rd Edition (1998) Addison Wesley Longman, Massachusetts.