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The number of units of enzyme in the test preparation can be calculated by

applying the Beer–Lambert law to calculate the amount of product formed per second:

enzyme units (katals per cm^3 test solutionÞ¼E^340

e

 a

1000

^1000

x

whereE 340 is the control-corrected change in the absorbance at 340 nm per second,

ais the total volume (cm^3 ) of reaction mixture (generally about 3 cm^3 ) in a cuvette

of 1 cm light path,xis the volume (mm^3 ) of test solution added to the reaction mixture

andelis the molar extinction coefficient for NADH at 340nm (6.3 103 M^1 cm^1 ). By

dividing the above equation by the total concentration of protein in the test enzyme

preparation, thespecific activity(katals kg^1 ) of the preparation can be calculated.

The scope of visible spectrophotometric enzyme assays can be extended by the use

of synthetic substrates that release a coloured product. Many such artificial substrates

are available commercially particularly for the assay of hydrolytic enzymes. The

favoured coloured products are phenolphthalein andp-nitrophenol both of which

are coloured in alkaline solution. An extension of this approach is the use of synthetic

dyes for the study of oxidoreductases. The oxidised and reduced forms of these

dyes have different colours. Examples are the tetrazolium dyes, methylene blue,

2,6-dichlorophenol indophenol and methyl and benzyl viologen.

Spectrofluorimetric methods

Fluorimetric enzyme assays have the significant practical advantage that they are

highly sensitive and can therefore detect and measure enzymes at low concentrations.

NAD(P)H is fluorescent and so enzymes utilising it can be assayed either by their

absorption at 340 nm or by their fluorescence (primary wavelength 340 nm, reference

wavelength 378 nm). Synthetic substrates that release a fluorescent product are also

available for the assay of some enzymes. An example is the assay ofb-D-glucuronidase

(EC 3:2:1:31) using 4-methylumbelliferyl-b-D-glucuronide as substrate and assaying

4-methyl-umbelliferone as the fluorescent product. The large commercial interest

in the development of inhibitors of kinases, phosphatases and proteases for their

therapeutic potential has stimulated the development of assays for these enzymes

using fluorogenic substrates to allow kinetic measurements of enzyme activity to be

undertaken in vivo using the principle of fluorescence resonance energy transfer

(FRET). These substrates contain two fluorochromes situated less than 100 A ̊apart

joined by a ‘linker’ that is cleaved by the test enzyme and such that the emission

wavelength of the donor fluorochrome overlaps with the excitation wavelength of the

acceptor fluorochrome allowing the former to transfer energy to the latter. The most

commonly used fluors are cyan fluorescent protein, red fluorescent protein and yellow

fluorescent protein.

Luminescence methods

Bioluminescence reactions are commonly used as the basis for an enzyme assay due to

their high sensitivity. The assay of luciferase is an example:

luciferinþATPþO 2 Ð

luciferase

oxyluciferinþAMPþPPiþCO 2 þlight

604 Enzymes