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assay. It is essential that the substrate and product do not absorb at the same

wavelength and that the Beer–Lambert law (Section 12.2.2) is obeyed for the chosen

analyte. A large number of common enzyme assays are based on the interconversion

of NAD(P)þand NAD(P)H. Both of these nucleotides absorb at 260 nm but only the

reduced form absorbs at 340 nm. Enzymes that do not involve this interconversion

can be assayed by means of acoupled reactionthat involves two enzyme reactions

linked by means of common intermediates. The assay of 6-phosphofructokinase (PFK)

(EC 2:7:1:11) coupled to fructose-bisphosphatase aldolase (FBPA) (EC 4.1.2.13)

and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (EC 1:2:1:12) illustrates

the principle:

D-fructose-6-phosphate

PFK+Mg2+

G3PDH+Pi

ATP ADP FBPA

NADH NAD+

D-fructose-1,6-bisphosphate

D-glyceraldehyde-

phosphate

glycerone

phosphate

1,3-bisphosphoglyceric +

acid

The assay mixture would containD-fructose-6-phosphate, ATP, Mg^2 þ, FBPA, G3PDH,

NADþand Piall in excess so that the reaction would go to completion and the rate of

reduction of NADþand the production of NADH and hence the increase in absorbance

at 340 nm, would be determined solely by the activity of PFK added to the reaction

mixture in a known volume of the test enzyme preparation. In principle there is no

limit to the number of reactions that can be coupled in this way provided that the

enzyme under investigation is always present in limiting amounts.

(a)

Minutes

Product produced (μmol)

1

t 0 t 1 10 t 2 20 30

2

3

4 units

2 units

1 unit

B A

C

(b)

Rate (μmol min

–1)

0.2

1

0.4

0.6

23 4

Units of enzyme

t 1

t 2

t 0

Fig. 15.11The importance of measuring the initial rate in the assay of an enzyme. (a) Time-dependent variation

in the concentration of products in the presence of 1, 2 and 4 units of enzyme; (b) variation of reaction rate

with enzyme concentration using true initial rate (v 0 ) and two fixed time assays (t 1 andt 2 ).

603 15.3 Analytical methods for the study of enzyme reactions