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than the corresponding spectrophotometric and fluorimetric assays, and a concen-

tration range of 10^9 to 10^12 M can be achieved. The NADPH assay is by a factor of

20 less sensitive than the NADH assay.

12.5 Circular dichroism spectroscopy

12.5.1 Principles

In Section 12.3.3 we have already seen that electromagnetic radiation oscillates in all

possible directions and that it is possible topreferentially select waves oscillating in a

single plane, as applied for fluorescence polarisation. The phenomenon first known as

mutarotation(described by Lowry in 1898) became manifest in due course as a special

property ofoptically activeisomers allowing the rotationof plane-polarised light.

Optically active isomers are compoundsof identical chemical composition and topology,

but whose mirror images cannot be superimposed; such compounds are calledchiral.

Linearly and circularly polarised light

Light is electromagnetic radiation where the electric vector (E) and the magnetic vector

(M) are perpendicular to each other. Each vector undergoes an oscillation as the light

Example 4ENZYMATIC CALCIUM MONITORING

Question Calcium signalling is a common mechanism, since the ion, once it enters the
cytoplasm, exerts allosteric regulatory affects on many enzymes and proteins. How
can intracellular calcium be monitored?

Answer The EF-hand protein aequorin fromAequoreaspecies (jellyfish) has been used for

determination of intracellular calcium concentrations. Despite the availability of

calcium-specific electrodes, this bioluminescence assay presents advantages due to

its high sensitivity to and specificity for calcium. Since the protein is non-toxic, has

a low leakage rate from cells and is not intracellularly compartmentalised, it is

ideally suited for usage in living cells. Its disadvantages are the scarcity, large

molecular size, consumption during the reaction and the non-linearity of the light

emission relative to calcium concentration. The reaction is further sensitive to the

chemical environment and the limited speed in which it can respond to rapid

changes in calcium concentration, for example influx and efflux in certain cell

types. The protein possesses a reflective yellow colour and is non-fluorescent in its

apo- (non-calcium-bound) state. In the calcium-bound form, the prosthetic group

coelenterazine, a molecule belonging to the luciferin family, is oxidised to

coelenteramide and CO 2. Upon relaxation to the ground state, blue light of 469nm

wavelength is emitted.

509 12.5 Circular dichroism spectroscopy