Circular dichroism

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Circular

dichroism
[1][2]

(CD) refers

to

the

differential
[3]

absorption

of

left

and

right circularly

polarized light.

This phenomenon was discovered by Jean-Baptiste Biot, Augustin Fresnel, and Aim It is exhibited in the absorption bands of optically
[4]

Cotton in the first half of the 19th century.

active chiral molecules. CD spectroscopy has a wide range of applications in many different fields. Most notably, UVCD is used to investigate the secondary structure of proteins. investigate charge-transfer transitions.
[5] [2]

UV/Vis CD is used to

Near-infrared CD is used to investigate geometric and electronic Vibrational circular dichroism, which uses light from

structureby probing metal dd transitions.

[4]

the infrared energy region, is used for structural studies of small organic molecules, and most recently proteins and DNA.
Contents
[hide]

1 Physical principles

o o

1.1 Circular polarization of light 1.2 Interaction of circularly polarized light with matter

1.2.1 Delta absorbance 1.2.2 Molar circular dichroism 1.2.3 Extrinsic effects on circular dichroism 1.2.4 Molar ellipticity 1.2.5 Mean residue ellipticity

2 Application to biological molecules 3 Experimental limitations 4 See also 5 References 6 External links

[edit]Physical [edit]Circular

principles

polarization of light

Main article: Circular polarization Electromagnetic radiation consists of an electric and magnetic field that oscillate perpendicular to one another and to the propagating direction.
[6]

While linearly polarized light occurs when the electric field

vector oscillates only in one plane and changes in magnitude, circularly polarized light occurs when the

electric field vector rotates about its propagation direction and retains constant magnitude. For left circularly polarized light (LCP) with propagation towards the observer, the electric vector rotates counterclockwise.
[2]

For right circularly polarized light (RCP), the electric vector rotates clockwise.

[edit]Interaction

of circularly polarized light with matter

When circularly polarized light passes through an absorbing optically active medium, the speeds between right and left polarizations differ (cL cR) as well as their wavelength (L R) and the extent to which they are absorbed (LR). Circular dichroism is the difference L- R. The electric field of a light beam
[4]

causes a linear displacement of charge when interacting with a molecule (electric dipole), whereas the magnetic field of it causes a circulation of charge (magnetic dipole). These two motions combined cause an excitation of an electron in a helical motion, which includes translationand rotation and their associated operators. The experimentally determined relationship between the rotational strength (R) of a sample and the is given by

The rotational strength has also been determined theoretically,

We see from these two equations that in order to have non-zero , the electric and magnetic dipole moment operators ( and ) must transform as the

same irreducible representation. Cn and Dn are the only point groups where this can occur, making only chiral molecules CD active. Simply put, since circularly polarized light itself is "chiral", it interacts differently with chiral molecules. That is, 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 of a selected wavelength are alternately radiated into a (chiral) sample. One of the two polarizations 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 after passing through the sample.
[edit]Delta absorbance

By definition,

where A (Delta Absorbance) is the difference between absorbance of left circularly polarized (LCP) and right circularly polarized (RCP) light (this is what is usually measured). A is a function ofwavelength, so for a measurement to be meaningful the wavelength at which it was performed must be known.
[edit]Molar circular dichroism

It can also be expressed, by applying Beer's law, as:

where L and R are the molar extinction coefficients for LCP and RCP light, C is the molar concentration l is the path length in centimeters (cm). Then

is the molar circular dichroism. This intrinsic property is what is usually meant by the circular dichroism of the substance. Since is a function of wavelength, a molar circular dichroism value () must specify the wavelength at which it is valid.
[edit]Extrinsic effects on circular dichroism

In many practical applications of circular dichroism (CD), as discussed below, the measured CD is not simply an intrinsic property of the molecule, but rather depends on the molecular conformation. In such a case the CD may also be a function of temperature, concentration, and the chemical environment, including solvents. In this case the reported CD value must also specify these other relevant factors in order to be meaningful.
[edit]Molar ellipticity

Although A is usually measured, for historical reasons most measurements are reported in degrees of ellipticity. Molar ellipticity is circular dichroism corrected for concentration. Molar circular dichroism and molar ellipticity, [], 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:

where ER and EL are the magnitudes of the electric field vectors of the right-circularly and left-circularly polarized light, respectively. When ER equals EL (when there is no

difference in the absorbance of right- and leftcircular polarized light), is 0 and the light is linearly polarized. When either ER or EL is equal to zero (when there is complete absorbance of the circular polarized light in one direction), is 45 and the light is circularly polarized. Generally, the circular dichroism effect is small, so tan as is small and can be

approximated

in radians. I, of

Since light is

the intensity or irradiance,

proportional to the square of the electric-field vector, the ellipticity becomes:

Then by substituting for I using Beer's law in natural logarithm form:

The ellipticity can now be written as:

Since expression

A << 1, can

this be

approximated by expanding the exponentials in a Taylor series to first-order and then discarding terms of A in comparison and converting radians to degrees: with unity from

The linear dependence of solute concentration and pathlength by is

removed

defining

molar ellipticity as,

Then the

combining last two

expression with Beer's molar becomes: law,

ellipticity

The units of molar ellipticity are historically (degcm /dm ol). calculate molar To
2

ellipticity, the sample concentratio n (g/L), cell pathlength (cm), and the molecular weight (g/mol) must be known. If the sample is a protein, the mean

residual weight (average molecular weight of the amino acids it contains) is used in place of molecular weight, essentially treating the the

protein as a solution of

amino acids.
[edit]Mean

residue ellipticity Methods estimating secondary for

structure polymers,

in

proteins and polypeptides in particular, often require that measured molar ellipticity spectrum be converted to a normalized value, specifically a value independent of polymer length. Mean residue ellipticity is the the

used for this purpose; it is simply measured molar ellipticity of the

the molecule divided the by

number

of monomer units (residues) in the molecule.

[edit]Applica

tion to biological molecules

In this phenomenon will exhibited absorption bands of be in general,

any optically active molec ule. As a

consequence , circular is

dichroism

exhibited by biological molecules, because of

theirdextrorot ary and levor otary compo nents. more important that a secondary structure will also impart a distinct CD to its respective molecules. Therefore, is Even

the alpha helix of proteins and thedouble helix of nucle ic acids have CD spectral

signatures representativ e of their

structures. The capacity of CD to give a representativ e structural

signature makes it a

powerful tool in modern

biochemistry with applications that can be found virtually every field of study. CD is closely related the optical rotatory dispersion (O RD) technique, to in

and generally considered

is

to be more advanced. CD is

measured in or near the absorption bands of the molecule interest, while can ORD be of

measured far from these

bands. CD's advantage is apparent the analysis. Structural elements are more clearly distinguished since recorded bands do not overlap extensively at particular their in data

wavelengths as they do in ORD. principle these two In

spectral measuremen ts can be

interconverte d through an integral transform (Kramers Kronig relation), if all the absorptions are included in the

measuremen ts. The far-UV

(ultraviolet) CD spectrum of can proteins reveal

important characteristic s of

their second ary structure. CD can spectra be

readily used to the estimate fraction

of a molecule that is in

thealphahelix conform

ation, the betasheet confor mation, the betaturn conform ation, some or other

(e.g. random coil) conformation .

[7][8]

These

fractional assignments place important constraints on possible secondary conformation s that the can CD in the

protein be in.

cannot,

general, say where the

alpha helices that are

detected are located within molecule even completely predict how the or

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

concentratio n denaturing agents, e.g. Guanidin ium hydrochlorid e or urea. In this can way it of

reveal

important thermodyna mic information about molecule (such as the

theenthalpy and Gibbs free energy of denaturation) that cannot

otherwise be easily obtained. Anyone attempting to study protein find CD a will a

valuable tool for that verifying the

protein is in its native

conformation before undertaking extensive and/or expensive experiments with it. Also, there are a number other for of uses CD

spectroscopy in protein

chemistry not related alpha-helix to

fraction estimation. The near-UV CD spectrum (>250 nm) of proteins provides information on the tertiary structure. The signals in

obtained the 300 nm region due to

250

are the

absorption, dipole orientation and the

nature of the surrounding environment of the

phenylalanin e, tyrosine, (or

cysteine

S-S disulfide bridges) and tryptophan a mino acids.

Unlike in farUV CD, the near-UV CD

spectrum cannot assigned any particular 3D structure. Rather, nearUV spectra provide structural information on the nature of prosthetic groups proteins, e.g., the in the CD be to

heme groups in hemoglobi n and cytoch rome c. Visible CD

spectroscopy is a very

powerful technique to study metal protein interactions and resolve individual d d electronic can

transitions as

separate bands. spectra the light are CD in

visible region only

produced when a metal ion is in a chiral environment, thus, free

metal ions in solution are

not detected. This has the advantage of only observing the protein-

bound metal, so pH

dependence and stoichiometri es readily obtained. Optical activity transition metal ion in are

complexes have been to

attributed

configuration

al, conformation al and the

vicinal effects. Klewpatinon d and Viles (2007) have produced set empirical rules predicting the appearance of visible CD spectra Cu Ni
2+

a of

for

for

and

2+

square-

planar complexes involving histidine and main-chain coordination. CD gives

less specific structural information than X-ray crystallograp hy and protei n NMR spectro scopy, for

example, which give both atomic

resolution data. However, CD spectroscopy is a quick

method that does not

require large amounts proteins extensive data processing. Thus CD can be used to survey a of or

large number of solvent co nditions, varying temp erature, pH, salinity, and the presence of various

cofactors. CD spectros copy is usually used to proteins study in

solution, and thus it

complements methods that study solid the state.

This is also a limitation, that in

many are

proteins

embedded in membrane sin their

native state, and solutions containing membrane structures are strongly scattering. CD sometimes measured in thin films.
[edit]

often

is