Spectroscopy 61

positions for the normal bases. This leads to their use in monitoring tRNA structure and interactions.
Nucleic acid polymers show dramatic hypochromism and hyperchromism. These are effects that arise
due to aggregation of chromophores. Quantum mechanical theory says that due to interactions between
the electric dipoles present in the chromophores, groups of chromophores, which aggregate together,
have different effects on the absorption spectrum. In a polynucleotide, if the bases are stacked one on
the other, as for example in the double helical structure, this leads to a diminution of the intensities
of absorption. The effect is most noticeable at the 260 nm band and is called hypochromism. If on the
other hand the bases are arranged in almost the same plane, i.e. in an end-to-end arrangement, the
absorption intensity is increased leading to hyperchromism (Figure 4.1). Hyperchromism is commonly
used to follow the denaturation behaviour or the melting behaviour of the DNA double helix. At
higher temperatures or at other disruptive conditions, when the base-base stacking is disturbed and
the double helix is denatured, the intensity of absorption at 260 nm increases.

4.3 Circular Dichroism (CD) and Optical Rotatory Dispersion (ORD)

Most biological molecules or systems are optically active; i.e. they rotate the plane of polarised light.
If the rotation is clockwise when seen looking towards the light source, it is said to be dextro-rotatory.
62 Biophysics

If the rotation is counter clockwise, it is levo-rotatory. For any compound, the extent of rotation
depends on the number of molecules in the path of the polarised light, or in other words, for solutions,
on the concentration and on the path length of the beam through it. It also depends on the wavelength
of the radiation and the temperature. Optical activity is quantified by the specific rotation

i.e. the specific rotation at a temperature t and for wavelength is the measured rotation
divided by the concentration c in grams per cubic centimetre and the light path d in centimetres.
Optical activity may also be quantified as the molar rotation

where M is the gram molecular weight of the substance. For polymers, it is common to use the mean
residual rotation

where is the mean residue molecular weight, i.e. the average molecular weight of the monomers.
Rather than the rotation at a single wavelength, measurement of the change in optical activity with
change in wavelength yields more useful structural information. This is called Optical Rotatory
Dispersion (ORD). A closely related phenomenon is Circular Dichroism (CD). Plane polarised light
can be resolved into two beams, each circularly polarised, but in opposite senses (Figure 4.2). The

refractive index for the right circularly polarised light, may be different from that for the left
circularly polarised light Similarly the absorbance for the right circularly polarised light may
be different from When and are unequal, each component will be retarded to a different
extent as it passes through the sample. This leads to a change in the phases of the two components
when they make up the plane-polarised light, leading to a rotation of the plane of polarisation. This
is in fact the theoretical basis for the phenomenon of optical activity. Since the refractive index is
always dependent on the wavelength, the angle of rotation is also wavelength-dependent. At a given
wavelength the angle of rotation

where d is the path length. or more commonly, the specific rotation can be plotted as a
function of wavelength (Figure 4.3). This is called the Optical Rotatory Dispersion (ORD) spectrum.
The difference in the absorption between the right and the left components does not introduce a phase
 Spectroscopy 63

difference between the two, but instead produces a difference in intensity, i.e. a difference in magnitude
of the wave. The resultant beam is no longer plane polarised but is elliptically polarised (Figure 4.4).
The difference in absorbance is called the circular dichroism or CD. The ellipticity of the
ellipse is also a measure of the differential absorbance, known as the ellipticity

where the absorbance A is defined as in equation (4.1) for a particular wavelength The molar
ellipticity is defined as

where M is the molecular weight, d is the path length in centimetres and c is the concentration in
64 Biophysics

grams/ml. is measured in degrees. A plot of the difference in the refractive index, i.e.
versus the wavelength is called the ORD curve and a plot of the difference in absorbance, i.e.
versus the wavelength is called the CD curve. As seen in Figure 4.5, the two phenomena are
related and together are called the Cotton effect. Both CD and ORD curves can be negative or positive
and go by the names ‘negative Cotton effect’ and ‘positive Cotton effect’ respectively. Rigorously, the
measurement of either CD or ORD implies the other. However, in practice, especially with the
availability of appropriate instrumentation, CD is the measurement of choice.

The CD spectrum of a polymer is different from that of a monomer, the difference reflecting the
three dimensional arrangement. CD, therefore, is immensely useful to study three-dimensional structures
of biopolymers such as proteins and nucleic acids. The transitions discussed previously
dominate the spectra, and in principle, quantum mechanical calculations of the CD of the biopolymers
can be performed. This would allow the interpretation of the spectrum in terms of the structure. This
however is an extremely complex task and, in practice, the existing optical activity data for polypeptides,
proteins and nucleic acids with known structure are used in conjunction with semi-empirical equations
to analyse unknown systems.
For proteins the aim is to analyse the CD spectrum to obtain secondary structural features such as
the percentage of regions or regions or random coil regions. The protein CD
spectrum is dominated by the polypeptide backbone chromophores and the effects of the side chains
are usually negligible. It can be considered as a linear combination of the spectra of each of the
regular secondary structural regions. One may thus write

where is the measured ellipticity and are the fractional compositions of

and random coil regions respectively, and and are the measured CD
of polypeptides in the conformation indicated. This equation can be used to estimate and for
an unknown protein, provided and are known. There are two methods of
obtaining this information; both of them giving equivalent results when applied to unknown proteins.
In the first method homopolypeptides that are known to possess one of the above secondary structures
are chosen and the CD spectrum is measured for each. These spectra are used as the basis for
calculating the fractional compositions. The other semi-empirical method obtains the basis values of
the and from the CD spectra of proteins of known three-dimensional structure.