J o h a n n Putter
The term peroxidase (POD) in its widest sense includes a group of specific enzymes such as NAD-POD,
NADP-POD, fatty acid-POD, cytochrome-POD and glutathione-POD as well as a group of very non-
specific enzymes from different sources, which are simply known as POD (donor: H 0 -oxidoreductase, 2 2
EC 1.11.1.7). Only the latter group will be considered in this chapter. POD catalyses the dehydrogenation
of a large number of organic compounds such as phenols and aromatic amines, hydroquinones and hydro-
quinoid amines, especially benzidine derivatives. In particular, should be mentioned o-cresol , o-toluidine , 1 2
o-tolidine , d i - o - a n i s i d i n e
16
, and some azo dyes derived from these .
14,17-19 14
With few exceptions the typical POD belonging to group 1.11.1.7 are haemoproteins . Although the 20
real POD reaction consists of the transfer of hydrogen from a donor to H 0 , there are examples of 2 2
+ 0 4 - NADPH -> S - O H + N A D P + O H " ) . On the other hand, apart from the true POD,
2
+
peroxidative reactions can be catalysed by catalase with certain substrates, e. g. phenols, alcohols and
several inorganic c o m p o u n d s , as well as by haemoglobin and some of its break-down products
27-30
with typical POD substrates . Finally, oxidases can in many cases react like POD and use H 0 as
31,32
2 2
a source of oxygen . Although in the classical POD reactions the specificity for the hydrogen donor is
33
very low, that for the peroxide is much higher. It appears that apart from H 0 , only compounds having 2 2
the group — O —OH, e. g. acetyl-, methyl- and ethyl-hydroperoxide can act as substrates . 34-36 37
Haemoprotein POD occur in animals, in higher plants, e.g. horse radish, pineapples, figs, potatoes,
legumes, corn, root vegetables, tobacco p l a n t s , in y e a s t s , in moulds and in bacteria .
6 , 3 8 - 4 8 37-49 50 20
The longest and the best studied is horse radish POD. In mammals POD occur in leucocytes , 20,51,52
milk , liver , spleen , uterus , salivary glands , stomach wall , intestinal mucosa , lung , thyroid
53 54 55 56 57 55 20 55
glands , etc. They have been found in mammalian cells in the supernatant, microsomal and mitochondrial
58
fractions.
POD can be determined by the decrease of H 0 or the hydrogen donor or the formation of the oxidized
2 2
compound . Usually the third method is employed, and many different substrates have been used.
37
Very accurate values are obtained with di-o-anisidine , but the use of this compound should be limited 14
because of its potential carcinogenic activity. The older method for the assay of POD with guaiacol is 3
c) As auxiliary enzyme in the determination of hydrogen peroxide , catalase , oxidases , glucose 60 61 62 18,19
,
galactose , etc. 63
e) In general biochemistry.
Principle
The POD reaction consists of 2 successive steps each involving 1 electron . A general equation for POD 65
catalysed reactions cannot be formulated because the course of the reaction depends on the type of
686 Enzyme Activities: Oxidoreductases
substrate. In the simplest case the same molecule is hydrogen donor for both steps. The equation of the
overall reaction is usually:
(1) H 0
2 2 + D H -522+ 2 H 0 + D
2 2
The intermediate DH is in many cases detectable . Often the intermediate product radicals can give
14
rise to complicated secondary reactions. An example of a complicated reaction is the peroxidative oxidation
of guaiacol. As the literature about the resulting dehydrogenation product is somewhat contradictory 66,67
and as probably more than one compound results from the reaction of guaiacol with H 0 and peroxidase ,
2 2
67
a definite stoichiometric formula cannot be given here. Possibly the nature of the reaction product depends
on the reaction conditions. Under the best conditions described below, a stoichiometric reaction proceeds
in so far as one mole H 0 oxidizes one mole guaiacol. In this contribution the resulting end product is
2 2
called GDHP (guaiacol dehydrogenation product). The rate of formation of GDHP in the guaiacol assay
is a measure of the POD activity and can be determined spectrophotometrically. The extinction coefficient
for GDHP at 436 nm is 6.39 cm. per /imole guaiacol oxidized or per μπιοίε H 0 consumed.
2
2 2
The kinetics of the various POD are not uniform ( e . g . ) . Generally it holds that for H 0 there is
68,69
2 2
a definable Michaelis constant which is dependent on the concentration of the donor. Whether a Mi
chaelis constant can be obtained for the donor is a matter of controversy . In any case, under
33,70,71
the usual conditions there is considerable dependence of the rate of the reaction on the concentration
of the donor, while it is hardly affected by changes in the H 0 concentration. In order to obtain a valid
2 2
and linear relationship between the measurements and the enzyme activity, it is necessary to express the
enzyme activity as a reciprocal of the time required for a fixed conversion (constant increase in extinction).
Even here it is possible to convert the results to international units. The rate of reaction for various donors
is different; POD from different sources differ in regard to relative reaction rates with different donors . 37
The expression of POD activity in μπιοίε substrate converted per min. is therefore not generally valid,
but only applies for a particular substrate and particular concentration of substrate.
The pH optima of different POD with guaiacol as hydrogen donor are not the same. However, as most
POD are active over a relatively broad range of pH, assay of activity at pH 7.0 is possible in practically
all cases. In addition, the temperature dependence of plant and animal POD is different; because of the
general applicability and the technical simplicity it is proposed that 25 °C should be used. Saturation
of enzyme with H 0 or with substrate is not achieved under our conditions; the concentrations given
2 2
The extinction maximum of GDHP is at 418 nm. At 436 nm the extinction is not essentially different;
measurement at this wavelength has the advantage that it can be carried out with the usual filter photo
meters. The brown colour of GDHP is not stable for long: in the first 10 min. after formation it decreases
by 4 - 5 % . For accurate measurements, it is therefore recommended to add sufficient enzyme so that
the time required for the reaction does not exceed 5 min.
Equipment
Reagents
1. Potassium dihydrogen phosphate, 3. Guaiacol, synthetic, cryst. D A B 6
KH P0 2 4 4. Hydrogen peroxide solution, 30% (w/v).
2. Dipotassium hydrogen phosphate,
Κ ΗΡ0 ·3Η 0
2 4 2
Preparation of Solutions
I. Phosphate buffer (0.1 M ; pH 7.0):
a) Dissolve 13.61 g. K H P 0 in doubly distilled water and make up to 1000 ml.; - b)
2 4
this solution should be 0.485 ( ± 0 . 0 2 0 ) at 240 nm and 1 cm. light path. If no UV-spectro-
photometer is available, the peroxide content of the solution can be determined by
titration with 0.01 Ν K M n 0 . 4
Stability of Solutions
The guaiacol solution is stable for at least 24 hr. in a cold room; in the frozen state it is stable for several
months. The stability of the dilute H 0 solution depends on various factors; it is best to determine
2 2
Procedure
Collection, Treatment and Stability of Sample
Collection of sample: It depends on the type of experimental material. Blood must be removed
from organs of experimental animals.
cell fractions (see p. 407). Suspensions of cell particles can generally be added directly to
the cuvette.
The stability of enzyme in sample: Depends on situation, but in any case it is recommended
that during the work up and storage of the sample it should be kept at 0 - 4 °C. If the stability
of the sample has not been specially studied, the assay should be carried out within 5 hr. of
collection of sample.
688 Enzyme Activities: Oxidoreductases
Assay System
Wavelength: Hg 436 nm; light path: 1 cm.; final volume: 3.18 ml.; temperature: 25 °C. Read
against distilled water instead of H 0 solution (III). Bring buffer solution (I) to 25 °C before
2 2
the assay.
During the measured time interval (i. e. to attain the extinction increase of 0.100) the mean
concentration for guaiacol is 0.3 mM and for H 0 is 0.1 mM. 2 2
Calculations
According to equation (8) from p. 313 with e 4 3 6 n m = 6.39 cm. ///mole and under the conditions described
2
here*:
„ . 3.18 χ 0.100 χ 1000 500 . r T T / 1
Precision of Method
The most accurate values are obtained when the time required is between 1 and 3 min. If the solution
contains 250 U/l. (t = 2 min.), the standard deviation is 2 U/l. The coefficient of variation is about 1.03%.
Normal Values
Information on the normal values for POD can be obtained from the references cited in the Introduction
under the section on occurrence of enzyme.
Sources of Error
Effects on POD activity in vivo: Hormones can sometimes affect POD activity in hormone-dependent
tissues (e. g . ) ; certain drugs can decrease POD activity. In certain cases of poisoning there is decreased
64
activity. Malignant tumours usually have lower POD activity than the organ of origin (e. g. ). 72
Cyanide inhibits all POD; this inhibition can sometimes be used to identify "true" POD activity.
Specificity of Method
For accurate assays it is important to ensure that removal of blood from the samples is as complete as
possible (exsanguination on decapitation of animals; perfusion of organs with buffer solution) because
of the POD activity of leucocytes and the pseudoperoxidative action of haemoglobin in erythrocytes.
As a large portion of POD is located in cell particles the total content of POD is only obtained after
extensive homogenization.
For histochemical detection of POD, which is important for the differentiation of white cells , refer to
59
References