ENZYME HISTOCHEMISTRY

 Enzymes are proteins which act as

biological catalyst
 Enzyme identification is a subdivision of
protein histochemistry where enzymatic
reaction with a suitable substrate
provides the basis of the techniques
ENZYME HISTOCHEMISTRY
Objectives & Applications
 Demonstration of enzyme activity within tissue

Function
 Localisation of enzymes within cells

 Distinguish cells based on specific high

enzyme activity
 Labels for molecular techniques

 Enzyme histochemistry is the localisation of

enzymes in tissue by their specific action on a

substrate
Diagnostic Applications
 Not widely applied to surgical and autopsy
material for diagnostic purposes
 Mainly due to total or partial loss of
enzymes which occur when a tissue is
routinely fixed and processed into paraffin
 Some enzyme methods can be usefully
applied to paraffin and acrylic resin
embedded sections
Diagnostic Applications
cont…
 A few enzymes are still identifiable in routinely
processed paraffin sections, e.g. chloroacetate
esterase
 Cryostat sections of fresh frozen material are
required for most enzyme histochemical techniques
 Current common uses of enzyme histochemistry in
surgical cellular pathology laboratory are as follows:
1. Skeletal muscle biopsy using oxidative enzymes
2. Rapid and easy detection of ganglia and nerves in
cases of suspected Hirschsprung’s disease using
non specific esterase
Diagnostic Applications
cont…
3. Demonstration of specific lactase or sucrase
deficiency in jejunal biopsies
4. Jejunal biopsies using acid and alkaline
phosphatase in cases of gluten disease
5. Demonstration of various white blood cells using
chloroacetate esterase
6. Demonstration of mast cells non specific
chloroacetate esterase
7. Miscellaneous diagnostic applications e.g. use of
acid phosphatase in identification of prostate
carcinoma
8. As an immunohistochemical marker
Tissue preparation in enzyme
techniques
 Enzymes are labile and their preservation is of
the utmost importance
 Most enzymes are rapidly inactivated by most
tissue preparation methods
 For this reason paraffin wax and plastic
embedded sections are almost entirely useless
for demonstrating enzyme activity
 A few enzymes are still identifiable in routinely
processed paraffin sections, e.g. chloroacetate
esterase and haemoglobin peroxidase
Tissue preparation in enzyme
techniques cont…
 Unfixed frozen (cryostat) sections are the more
usual mode of preservation
 Fixation can sometimes be useful to stop
enzyme diffusion e.g. in hydrolytic enzymes
 This can be done either on the block before
sectioning or immediately after cutting when
the section can be rapidly fixed
 Fixing the section rather than the tissue is
often better since the length of fixation can be
very brief as penetration can be rapid in case
of sections
Tissue preparation in enzyme
techniques cont…
 For electron microscopy the staining is done
on the fresh tissue before processing (‘’block
staining’’)
 This means that only one method can
performed on each block of tissue and a
separate block is needed for each enzyme
being studied
 For the demonstration of hydrolytic enzymes
histochemical methods are usually applied to
sections prefixed in cold (4oC) formal calcium
Classification of enzymes
1. Oxidoreductases
 Largest and important group in enzyme
histochemistry
 Are enzymes that affect their substrates by
changing their redox potential and often
transfer electrons between the substrates
 Three types are considered:
a) Oxidases
 Catalyse oxidation of a substrate in the
presence of oxygen
Oxidoreductases
b) Peroxidases
 Catalyse oxidation of a substrate by removing
hydrogen which combines with hydrogen peroxide
c) Dehydrogenases
 Which involve the transfer of hydrogen from a
substrate to a carrier molecule
Other important groups:
1. Transferases
 These enzymes catalyse the transfer of the
radicals of two compounds without the loss or
uptake of water
2. Hydrolases
 Catalyse the introduction of water or its
elements into specific substrate bonds,
These enzymes include:
 Esterases
 Lipases
 Phosphatases
 Glycosidases
 Peptidases
 pyrophosphatases
Factors affecting enzyme
activity
1. Temperature
 Optimal temperature for the majority of
enzyme reactions is 37oC
 Enzymes are rapidly denatured at higher
due to their protein content
 Lower temperatures between 4 and 30oC
can be usefully employed in histochemistry
 This is because the rate of the enzymic
reaction is slowed and in case of active
enzymes, abetter localisation demonstrated
pH
 Majority of enzymes have a pH at
which the rate of the reaction in
optimal
 For a large number of enzymes this
is pH 7.0 – 7.2
 Alkaline phosphatase at pH 9.2 and
acid phosphatase at pH 5.0 are
examples of exceptions to this rule
Inhibitors
 Enzyme activity in sections can be destroyed
by using chemical substances known as
inhibitors
 There are three main types of inhibitors:
1. Specific inhibitors, e.g. eserine for the
cholinesterases
2. Non specific inhibitors, e.g. heat for all
enzymes
3. Competitive inhibitors, e.g. chemical which
compete with the substrate for active
enzyme sites
Types of histochemical
reactions
 There are four main types of
histochemical reactions available for
the demonstration of enzymes:
1. Simultaneous coupling (capture) using
diazonium salts
2. Post - incubation coupling using
diazonium salts
3. Metal precipitation techniques
4. Self - coloured substrate
Simultaneous coupling
(capture) using diazonium
salts
 Incubation mixture containing a substrate
and a diazonium salt are applied to
suitable sections in a buffered solution
 Enzyme present in the section hydrolyses
the substrate to form an invisible
primary reaction product (PRP)
 This complex couples with the diazonium
salt to produce the final reaction
product (FRP) which is visible, often
coloured deposit under the microscope
Simultaneous coupling
(capture) using diazonium
salts cont…
 This type of method by the azo dye
methods for phosphatases
 The PRP is colourless where as the
FRP is coloured
 The substrate must be soluble in
water or in the buffer medium so
that there is sufficient substrate
available for the enzyme to
hydrolyse
Simultaneous coupling
(capture) using diazonium
salts cont…
 The histochemical method must be
performed at a pH at which the
enzyme shows maximal activity,
usually pH 7.0 for most enzymes
 The diazonium salt also has an
optimal pH for its most efficient
rate of coupling, this should also
be considered
Simultaneous coupling
(capture) using diazonium
salts cont…
 The PRP must be fairly insoluble, if too
soluble then not all of it will couple with
the diazonium salt
 The quantity of both substrate and
diazonium salt must be correct, too
much of either will cause inhibition of
the rate of hydrolysis of the substrate
on one hand, and inhibition of the
formation of PRP on the other
Simultaneous coupling
(capture) using diazonium
salts cont…
Post - incubation coupling
using diazonium salts
 In this reaction the enzyme hydrolyses
the substrate and produces a reasonably
insoluble PRP
 The subsequent coupling is carried out in
a separate solution
 This type of method relies upon the PRP
remaining at the initial site of hydrolysis
 It is important that diffusion of the PRP
does not occur during the coupling
process
Post - incubation coupling
using diazonium salts
 It is important that diffusion of the PRP does not occur
during the coupling process
Advantages of pot-incubation coupling:
1. The optimal pH for the initial substrate enzyme
reaction can be attained in the first incubation, say pH
7.0 and possibly different optimal pH for the coupling
can be produced in the second coupling stage, e.g. 9.1
2. The method avoids the deleterious effects
that diazonium salts may have when the
incubation time is long
3. Long exposure of tissue to diazonium salts in acid or
alkaline solutions produces non specific staining
Post incubation coupling
using diazonium salts cont…
 Draw backs of of this method are:
1. Solubility of the PRP leading to
excessive diffusion between the
two stages
 Localisation may be poor unless
the PRP is extremely insoluble
Metal precipitation technique
 Commonly applied to the demonstration of
phosphatases
 The phosphate ions released as a result of
enzyme activity on the substrate combine
with a suitable metallic cation to produce an
insoluble precipitate of metal phosphate
 The phosphates so produced are invisible
but can be rendered visible by converting
them to black sulphides by treatment with
ammonium sulphide
Metal precipitation
techniques
 Metallic cations used for combining
with the released phosphate are
calcium and lead
 This method may be regarded as
simultaneous coupling, but using
metallic ions instead of diazonium
salts
Self - coloured substrate
 Involves the use of a coloured substrate that
is also soluble
 During hydrolysis the enzyme removes the
soluble grouping without affecting the colour
 The PRP is therefore coloured and insoluble,
and no coupling stage is necessary
 The insoluble coloured reaction product is
precipitated at the site of enzyme activity
Diazonium salts
 Are prepared by treating primary
aromatic amines with an acid solution of
sodium nitrate
 The resulting salt, generally a chloride,
will react readily with phenols or with
aryl amines to produce the
corresponding intensely coloured
insoluble azo dye, but a few are dyes,
e.g. pararosanilin and fast garnet GBC
Diazonium salts
 The salts themselves are usually colourless
 Diazonium salts can not be kept indefinitely
 Must be stored in a cool dark place and
renewed at six month intervals
 The solubility of diazonium salts is limited,
especially in the alkaline pH range
Hydrolytic enzymes – The
phosphatases
 Are enzymes capable of hydrolysis
organic phosphate esters
 Classified on the basis of their
optimal pH levels – those that exhibit
maximum activity at around pH 9.0
are termed alkaline phosphatases
 Those whose peak is around pH 5.0
are called acid phosphatases
Hydrolytic enzymes – The
phosphatases
 These enzymes are non specific as they
hydrolyse a wide range of organic
phosphate esters
 The exception is glucose -6-
phosphatase which only
dephosphorylate glucose-6-phosphate
at pH 6.5
 This is there considered a specific
phosphatase
Alkaline phosphatase
 Enzyme is localised in cell membranes in
the kidney and many other tissue
 Exhibits maximum activity at alkaline pH,
in the region of 9.0 – 9.6
 They are activated by magnesium,
manganese and cobalt ions
 Cyanide and cysteine inhibit alkaline
phosphatase activity and may be
incorporated in the incubating medium to
provide a control
Alkaline phosphatase
 There two types of histochemical methods available
for the demonstration of alkaline phosphatases. They
are:
 Gomori's calcium phosphate method (metal
substitution)
 Azo dye coupling methods using either simultaneous
coupling or post coupling
 The section is placed in an incubating solution
containing a substrate (sodium β-glycerol
phosphate), and calcium ions (calcium nitrate) plus
an activator for the phosphate (magnesium chloride),
a precipitate calcium phosphate is formed at the
site of enzyme activity
Gomori’s calcium phosphate
(Metal precipitation) method
 The alkaline phosphatase liberates
phosphate ions from sodium β-glycerol
phosphate and this combines with calcium
ions to form calcium phosphate
 This precipitate is in turn treated with cobalt nitrate
to produce a precipitate of cobalt phosphate
 This reaction product can not be seen with light
microscope
 Therefore this precipitate is treated with dilute
solution of ammonium sulphide to produce a
visible black precipitate of cobalt sulphide
Gomori’s calcium phosphate
method – Summary of
principle
Gomori’s calcium phosphate
method
Gomori’s calcium phosphate
method
Gomori’s calcium phosphate
method
Alkaline Phosphatase azo dye
coupling method using α -
naphthol phosphate

 Are also simultaneous coupling

techniques
 The method employs α - naphthol
phosphate as the substrate, together
with a fresh diazonium salt (Fast red TR
salt, Fast blue RR salt)
 The incubating medium is buffered to pH
9.2
Alkaline Phosphatase azo dye
coupling method using α -
naphthol phosphate
 The enzyme liberates α-naphthol (PRP) from
the substrate
 This is subsequently coupled to a diazonium
salt to produce an insoluble azo dye at the
site of enzyme activity
 Best results are obtained when coupling
between the α - naphthol and the diazonium
salt occurs rapidly
 This depends on the choice of diazonium salt
and upon the pH of the incubating solution
Alkaline Phosphatase azo dye
coupling method using α -
naphthol phosphate
Acid phosphatase
 The enzymes are distributed widely through
out the body; the kidney, liver and spleen
 Fluorides ions inhibit these enzymes, and
the inclusion of sodium fluoride in the
incubating medium affords a reliable control
measure
 As with alkaline phosphatase, there are two
reliable techniques for the demonstration of
these enzymes: the Gomori lead phosphate
method and the azo-dye coupling methods
using simultaneous coupling
Gomori lead phosphate
method
 The section is incubated in a medium
containing sodium-β-phosphate as the
substrate and lead nitrate; no activator is
required
NOTE:
 Calcium phosphate, which is formed at the

site of activity in the alkaline phosphatase

technique can not be used in the instance
because of its solubility at acid pH levels
Gomori lead phosphate
method cont…
 The enzyme splits phosphate ions from
substrate and these form an insoluble
precipitate of lead phosphate at the site
of enzyme activity
 The sections are treated with a dilute
solution of ammonium sulphide and the
precipitate converted to lead sulphide
 This is visible under the microscope as a
dark brown, granular deposit
Gomori lead phosphate
method cont…
Gomori lead phosphate
method cont…
Gomori lead phosphate
method cont…
Azo dye simultaneous
coupling method
 The substrate used is a sodium α-naphthyl
phosphate (1-naphthyl phosphoric acid) dissolved
in 0.1 M veronal acetate buffer at pH 5.0
 α – naphthol is released at the site of enzyme
activity and coupled with a suitable diazonium salt
 The problem with azo – dye methods for the
demonstration of acid phosphatase is to find a
diazonium salt that will couple satisfactorily at a
low pH
 Of all the comprehensive diazonium salts studied
it was concluded that Fast Garnet GBC gave the
best results
Acid phosphatase azo-dye
coupling method simultaneous
Coupling
Acid phosphatase azo-dye
coupling method simultaneous
Coupling cont…
Esterases
 Are enzymes capable of
hydrolysing carboxylic acid
 There are many types of esterases,
acting upon a number of
substrates
 Most of the enzymes have an
optimal pH between 5.0 and 9.0
Esterases cont…
 Each enzyme is capable of hydrolysing
a number of different substrates, and
many enzymes are capable of
hydrolysing the same substrates
 The majority of esterase enzymes are
able to hydrolyse α-naphthyl acetate
as a substrate
 These enzymes are called non-specific
esterases
Esterases cont…
 Majority of specific esterases are
also capable of hydrolysing a
simple ester such as a α-naphthyl
acetate
 There are three groups of non
specific esterases:
1. Carboxylesterases
Esterases cont…
1. Arylesterases
2. Acetylesterases
 Specific esterases are subdivided
as follows:
1. Acetylcholinesterases
2. cholinesterases
3. lipases
Esterase demonstration in
paraffin sections
Chloroacetate esterase
 This esterase is capable of resisting

the effects of paraffin processing and

can be demonstrated in paraffin
sections
 Is used to demonstrate mast cells and

white cells of the myeloid series

 The pH of the incubating medium

must be below 7.1

Esterase demonstration in
paraffin sections cont…
 The incubation time varies according to
the type of sections used; frozen sections
show strong activity after 5 minutes, while
paraffin wax will require a longer time
 Mast cells will stain rapidly while myeloid
cells, if in paraffin wax sections, may take
several hours
 Temperature of the water bath should be
kept below 37oC
Chloroacetate esterase
method cont…
Dehydrogenases
 Are enzymes which have the ability to
remove hydrogen from the substrate
and transfer to another substance
 The substance which acts as a hydrogen
acceptor is either nicotinamide adenine
dinucleotide phosphate (NAD), or
nicotinamide adenine dinucleotide
phosphate (NADP), or aflavoprotein
Dehydrogenases cont…
 NAD and NADP are also known as
co-enzymes 1 and 2 respectively
(See table)
 Because of the ability of
dehydrogenases to remove
hydrogen from the substrate, they
era regarded as oxidative enzymes
(See diagram)
Clinical name/Standard
abbreviation
Reduction path way for
tetrazolium salts
Dehydrogenases
 When they have accepted the hydrogen
released by the action of the
dehydrogenase, they are known as
reduced NAD and NADP, signified as
NADH and NADPH (See table)
 In some instances , the dehydrogenase
itself can act as hydrogen acceptor, and
is then reduced by flavoproteins in a
subsequent reaction
Dehydrogenases cont…
 The ‘diaphorases’ are dihydrogenases
which catalyse the dehydrogenation of
the reduced forms of NAD and NADP,
that is, they catalyse the reactions
 NADH → NAD + H
 NADPH → NADP + H
 The dehydrogenases are mitochondrial
enzymes and will be removed by
standard fixation techniques
Dehydrogenase Reaction
catalysed
Rationale of demonstration of
dehydrogenases cont…
 Dehydrogenase activity is
demonstrated histochemically using
tetrazolium salts
 These salts are colourless and
water soluble
 Are able to accept hydrogen from
the substrate by the enzyme action
Rationale of demonstration of
dehydrogenases cont…
 Tetrazolium salts which have been
reduced in this way produce an insoluble,
highly coloured, microcrystalline deposit
of a formazan compound
 An unfixed frozen section is incubated in
a medium containing the specific
substrate, the tetrazolium salt, buffer,
the co- enzyme (if required) and any
activators and chelators necessary
Types of tetrazolium salts
 Two main types are used, the
monotetrazolium salts and
ditetrazolium salts
 At the present time, salts from
each of the two groups are in
routine use for dehydrogenase
methods
Types of tetrazolium salts
cont…
 Ditetrazolium chloride –nitro-BT was
introduced for the demonstration of
succinic dehydrogenase
 The furmazan produced with this salt is
highly coloured, microcrystalline and
insoluble in lipid
 An example of the monotetrazole is
3(4;5-dimethylthiazolyl-2): 5-diphenyl
tetrazolium bromide (MTT)
Rationale of demonstration of
dehydrogenases