SEMINAR

ON
CIRCULAR DICHROISM
SUBMITTED TO SUBMITTED BY
MR. MANOJ KUMAR SURABHI ANAND
LECTURER M.PHARM[PHARMACOGNOSY]
DEPARTMENT OF PHARMACY 1ST YEAR
SITM,LUCKNOW
 Circular Dichroism

Circular dichroism (CD) spectroscopy measures differences in the absorption of

left-handed polarized light versus right-handed polarized light.

"Dichroism" is used to denote direction-dependent light absorption.

 Light
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 Polarization of Light
In ordinary light wave the vibration of electric vector occur in all possible
direction in a plane perpendicular to direction of propagation of wave

When light pass through polarizer the vibration of electric vector occur only
in a single in plane perpendicular to direction of propagation

Direction of propagation
 Types of polarized light

Plane polarized light consists two circularly polarized components of

equal intensity

Two circularly polarized components are like left- and right-handed

springs

As observed by looking at the source, right-handed circularly polarized

light rotates clockwise

Frequency of rotation is related to the frequency of the light.

 Circular Polarized Light

• Linearly polarized light:

Electric vector direction constant - magnitude varies.

• Circularly polarized light:

Electric vector direction varies - magnitude constant.

 CircularlyPolarizedLight
Circular polarized light:
Circular polarized light:

Electric vector direction varies - magnitude constant

Electric vector direction varies - magnitude constant

So its in two forms: left and right handed

So its in two forms: left and right handed
TYPES OF CIRCULAR DICHROISM
1.Far-UV Circular Dichroism (190-240nm):

“Far-UV” protein spectra are sensitive to secondary structure.

2.Near-UV Circular Dichroism (240-340nm):

“Near-UV” protein spectra are sensitive to tertiary structure.

 Plane-Polarized Waves in an Absorbing Medium

The following animation shows what happens when a plane-polarized wave traverses
a medium that absorbs light but does not refract it
Circularly Polarized Waves in an Absorbing Medium

This animation shows what happens when a circularly polarized

light wave passes throughout a light absorbing medium
 Circular Dichroism (CD) Spectroscopy
CD is observed when optically active matter absorbs
left and right hand circular polarized light

All optically active compounds exhibit CD

The CD is a function of wavelength. CD spectra for distinct

types of secondary structure present in peptides and proteins
are different.

The analysis of CD spectra can therefore yield valuable

information about secondary structure of biological
macromolecules.
 Principle of CD

Chiral molecules produce a CD spectrum because they absorb left and right handed
polarized light to different extents and thus are considered to be "optically active“

CD is measured as a quantity called mean residue ellipticity (degrees-cm2/dmol)

 Dichroism

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Instrumentation - Lab-Based
Spectropolarimeter
 Nitrogen Purging

The function of purging the CD instrument with nitrogen is to remove

oxygen from the lamp housing, monochromator, and the sample chamber.

The reason for removing oxygen is that oxygen absorbs deep UV light, thus
reducing the light available for the measurement.
The CD Spectropolarimeter
 Sample Preparation

Additives, buffers and stabilizing compounds: Any compound which absorbs

in the region of interest (250 - 190 nm) should be avoided.

A buffer or detergent or other chemical should not be used unless it can be

shown that the compound in question will not mask the protein signal.
 Sample Preparation

Protein solution: From the above follows that the protein solution should
contain only those chemicals necessary to maintain protein stability, and
at the lowest concentrations possible. Avoid any chemical that is
unnecessary for protein stability/solubility. The protein itself should be as
pure as possible, any additional protein or peptide will contribute to the
CD signal.
 Sample Preparation

Contaminants: Particulate matter

anything that adds artificial signal
contributions) to the CD spectrum must be
avoided
 Typical Initial Concentrations

Protein Concentration: 0.5 mg/ml

Need very little sample: 0.1 mg

Cell Path Length: 0.5 mm

Stabilizers (Metal ions, etc.): minimum

Buffer Concentration : 5 mM or as low as possible

 Why use CD?
Simple and quick experiments

No extensive preparation

Measurements on solution phase

Relatively low concentrations/amounts of sample

Microsecond time resolution

Any size of macromolecule

 Advantages
Simple and quick experiments

No extensive preparation

Measurements on solution phase

Relatively low concentrations/amounts of sample

Microsecond time resolution

Any size of macromolecule

Drawbacks to CD spectral analysis

Difficult to quantitate similarity or

differences

Certain buffer components absorb strongly

in Far-UV & can cause interference
 Applications of CD

Determination of secondary structure of proteins that cannot be crystallized

Investigation of the effect of e.g. drug binding on protein secondary structure

Studies of the effects of environment on protein structure

Study of ligand-induced conformational changes

 CONCLUSION
CD is a useful method for looking at secondary structures
of proteins and peptides.
It is an adaptation of standard absorption spectroscopy in which the difference
in the abs between left and right hand circularly polarized light is measured.

CD can be measured under a wide range of conditions -

e.g., good for membrane proteins.

CD can be used to measure change .

CD compliments other more detailed techniques such as

crystallography .
 Thankyou
& Questions