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Further applications include the use of circular dichroism for an observable for

kinetic measurements using the stopped flow technique (see Section 15.3).

12.6 Light scattering

The scattering of light can yield a number of valuable insights into the properties of

macromolecules, including the molecular mass, dimensions and diffusion coefficients,

as well as association/dissociation properties and internal dynamics. The incident

light hitting a macromolecule is scattered into all directions with the intensity of

the scatter being only about 10^5 of the original intensity. The scattered light is

measured at angles higher than 0and less than 180. Most of the scattered light

possesses the same wavelength as the incident light; this phenomenon is calledelastic

light scattering. When the scattered light has a wavelength higher or lower than the

incident light, the phenomenon is calledinelastic light scattering. The special prop-

erties of lasers (see Section 12.1.3) with high monochromaticity, narrow focus and

strong intensity, make them ideally suited for light scattering applications.

12.6.1 Elastic (static) light scattering

Elastic light scattering is also known asRayleigh scatteringand involves measuring

the intensity of light scattered by a solution at an angle relative to the incident laser

beam. The scattering intensity of macromolecules is proportional to the squared

Example 5DETERMINATION OF THE SECONDARY STRUCTURE CONTENT
OF A PROTEIN SAMPLE

Question You have purified a recombinant protein and wonder whether it adopts a folded
structure. How might you address this problem?

Answer CD spectra of poly-L-amino acids as well as proteins with known three-dimensional

structure have been obtained and are used as standards for deducing the secondary

structure composition of unknown proteins. The simplest approach is a visual

comparison of the shape of the CD spectrum with the three ‘Fasman standard spectra’

(Fig. 12.18), allowing conclusions as toahelix,bstrand and random coil structure.

CD deconvolution is a curve-fitting process where the experimental CD spectrum

is fitted with a given set of basis spectra using a weighting scheme. The estimated

weighting coefficients determined in the fitting process reveal the percentage of each

form of secondary structure in proteins. Different algorithms for deconvolution have

been generated, ranging from a simple linear combination of three or five basis sets,

to fitting procedures using 10–30 basis spectra and different mathematical

algorithms (CONTIN fit, neural networks, etc.).

514 Spectroscopic techniques: I Photometric techniques