Page 111 - Color Atlas of Biochemistry

P. 111

102 Metabolism

Enzymatic analysis absorption is recorded at a constant
wavelength of 340 nm. The uncatalyzed LDH
Enzymes play an important role in biochem- reaction is very slow. It is only after addition
ical analysis. In biological material—e. g., in of theenzymethat measurablequantities of
body fluids—even tiny quantities of an en- NADH are formed and absorption increases.
zyme can be detected by measuring its cata- Since according to the Beer–Lambert law the
lytic activity. However, enzymes are also used rate of the increase in absorption ∆A/∆tis
as reagents to determine the concentrations
of metabolites—e. g., the blood glucose level proportional to the reaction rate ∆c/∆t. The
absorption coef cient ε at 340 nm or compar-
(C). Most enzymaticanalysisproceduresuse ison with a standard solution can be used to
the method of spectrophotometry (A).
calculate LDH activity.

A. Principle of spectrophotometry C. Enzymatic determination of glucose
Many substances absorb light in the visible or Most biomolecules do not show any absorp-
ultraviolet region of the spectrum. This prop- tion in the visible or ultraviolet spectrum. In
erty can be used to determine the concentra- addition, they are usually present in the form
tion of such a substance. The extent of light of mixtures with other—similar—compounds
absorption depends on the type and concen- that would also react to a chemical test pro-
tration of the substance and on the wave- cedure. These two problems can be avoided
length of the light used. Monochromatic by using an appropriate enzyme to produce a
light—i. e., light with a defined wavelength colored dye selectively from the metabolite
isolated from white light using a monochrom- that is being analyzed. The absorption of the
ator—is therefore used. Monochromatic light dye canthenbe measured.
with an intensity of I 0 is passed through a Aprocedure (1)thatis often used to mea-
rectangular vessel made of glass or quartz (a sure glucose when monitoring blood glucose
cuvet), which contains a solution of the ab- levels (see p. 160) involves two successive re-
sorbing substance. The absorption A of the actions. The glucose-specific enzyme glucose
solution (often also referred to as its extinc- oxidase (obtained from fungi) first produces
tion)is defined as the negative decadic loga- hydrogen peroxide, H 2 O 2 , which in the second
rithm of the quotient I/I 0 . The Beer–Lambert step—catalyzed by a peroxidase—oxidizes a
law states that A is proportional to the con- colorless precursor into a green dye (2).
centration c of the absorbing substance and When all of the glucose in the sample has
the thickness d of the solution it passes been used up, the amount of dye formed—
through. As mentioned earlier, the absorption which can be measured on the basis of its
coef•cient ε depends on the type of substance light absorption—is equivalent to the quantity
and the wavelength. of glucose originally present.

B. Measurement of lactate dehydrogenase
activity
Measurement of lactate dehydrogenase (LDH)
activity takes advantage of the fact that while
+
the reduced coenzyme NADH + H absorbs
+
lightat340 nm,oxidized NAD does not. Ab-
sorption spectra (i. e., plots of A against the
wavelength) for the substrates and the coen-
zymes of the LDH reaction are shown in Fig. 1.
Differences in absorption behavior between
+
NAD and NADH between 300 and 400 nm
result from changes in the nicotinamide ring
during oxidation or reduction (see p. 32). To
measure the activity, a solution containing
+
lactate and NAD is placed in a cuvet, and

106 107 108 109 110 111 112 113 114 115 116