Zymography is an
electrophoretic
Electrophoresis, from Ancient Greek ἤλεκτρον (ḗlektron, "amber") and φόρησις (phórēsis, "the act of bearing"), is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric fie ...
technique for the detection of
hydrolytic
Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution, elimination, and solvation reactions in which water is the nucleophile.
Biological hydrolysi ...
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
s, based on the substrate repertoire of the enzyme. Three types of zymography are used; ''in gel'' zymography, ''in situ'' zymography and ''in vivo'' zymography
For instance,
gelatin
Gelatin or gelatine (from la, gelatus meaning "stiff" or "frozen") is a translucent, colorless, flavorless food ingredient, commonly derived from collagen taken from animal body parts. It is brittle when dry and rubbery when moist. It may also ...
embedded in a
polyacrylamide gel
Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in biochemistry, forensic chemistry, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their ...
will be digested by active
gelatinase Gelatinases are enzymes capable of degrading gelatin.
Gelatinases are expressed in several bacteria including ''Pseudomonas aeruginosa'' and ''Serratia marcescens''.
In humans, the gelatinases are matrix metalloproteinases MMP2 and MMP9
Matrix ...
s run through the gel. After
Coomassie staining, areas of degradation are visible as clear bands against a darkly stained background.
Modern usage of the term zymography has been adapted to define the study and cataloging of fermented products, such as beer or wine, often by specific brewers or winemakers or within an identified category of fermentation such as with a particular strain of yeast or species of bacteria.
Zymography also refers to a collection of related, fermented products, considered as a body of work. For example, all of the beers produced by a particular brewery could collectively be referred to as its zymography.
See also
Zymology
Zymology, also known as zymurgy,, "the workings of fermentation". is an applied science that studies the biochemical process of fermentation and its practical uses. Common topics include the selection of fermenting yeast and bacteria species an ...
or the applied science of zymography. Zymology relates to the biochemical processes of fermentation, especially the selection of fermenting yeast and bacteria in brewing, winemaking, and other fermented foods. For example, beer-making involves the application of top (ale) or bottom fermenting yeast (lager), to produce the desired variety of beer. The synthesis of the yeast can impact the flavor profile of the beer, i.e. diacetyl (taste or aroma of buttery, butterscotch).
Gel zymography
Samples are prepared in a standard, non-reducing loading buffer for
SDS-PAGE
SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular masses between 5 and 250 kDa. T ...
. No reducing agent or boiling are necessary since these would interfere with refolding of the enzyme. A suitable substrate (e.g.
gelatin
Gelatin or gelatine (from la, gelatus meaning "stiff" or "frozen") is a translucent, colorless, flavorless food ingredient, commonly derived from collagen taken from animal body parts. It is brittle when dry and rubbery when moist. It may also ...
or
casein
Casein ( , from Latin ''caseus'' "cheese") is a family of related phosphoproteins (CSN1S1, αS1, aS2, CSN2, β, K-casein, κ) that are commonly found in mammalian milk, comprising about 80% of the proteins in cow's milk and between 20% and 60% of ...
for protease detection) is embedded in the resolving gel during preparation of the
acrylamide gel. Following
electrophoresis
Electrophoresis, from Ancient Greek ἤλεκτρον (ḗlektron, "amber") and φόρησις (phórēsis, "the act of bearing"), is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric fie ...
, the
SDS is removed from the gel (or zymogram) by incubation in unbuffered
Triton X-100
Triton X-100 (''n'') is a nonionic surfactant that has a hydrophilic polyethylene oxide chain (on average it has 9.5 ethylene oxide units) and an aromatic hydrocarbon lipophilic or hydrophobic group. The hydrocarbon group is a 4-( 1,1,3,3-tetramet ...
, followed by incubation in an appropriate digestion buffer, for an optimized length of time at 37 °C. The zymogram is subsequently stained (commonly with
Amido Black
Amido black 10B is an amino acid staining azo dye used in biochemical research to stain for total protein on transferred membrane blots, such as the western blot. It is also used in criminal investigations to detect blood present with latent fi ...
or
Coomassie brilliant blue
Coomassie brilliant blue is the name of two similar triphenylmethane dyes that were developed for use in the textile industry but are now commonly used for staining proteins in analytical biochemistry. Coomassie brilliant blue G-250 differs from ...
), and areas of digestion appear as clear bands against a darkly stained background where the substrate has been degraded by the enzyme.
Variations on the standard protocol
The standard protocol may require modifications depending on the sample enzyme; for instance, ''D. melanogaster'' digestive
glycosidases
Glycoside hydrolases (also called glycosidases or glycosyl hydrolases) catalyze
Catalysis () is the process of increasing the rate of a chemical reaction by adding a substance known as a catalyst (). Catalysts are not consumed in the rea ...
generally survive reducing conditions (i.e. the presence of 2-mercaptoethanol or
DTT), and to an extent, heating. Indeed, the separations following heating to 50 °C tend to exhibit a substantial increase in band resolution, without appreciable loss of activity.
A common protocol used in the past for zymography of α-amylase activity was the so-called starch film protocol of W.W. Doane. Here a native PAGE gel was run to separate the proteins in a homogenate. Subsequently, a thin gel with starch dissolved (or more properly, suspended) in it was overlaid for a period of time on top of the original gel. The starch was then stained with Lugol's iodine.
Gel zymography is often used for the detection and analysis of enzymes produced by microorganisms.
This has led to variations on the standard protocol e.g. mixed-substrate zymography.
Reverse zymography copolymerizes both the substrate and the enzyme with the acrylamide, and is useful for the demonstration of
enzyme inhibitor
An enzyme inhibitor is a molecule that binds to an enzyme and blocks its activity. Enzymes are proteins that speed up chemical reactions necessary for life, in which substrate molecules are converted into products. An enzyme facilitates a sp ...
activity. Following staining, areas of inhibition are visualized as dark bands against a clear (or lightly stained) background.
In imprint technique, the enzyme is separated by native
gel electrophoresis
Gel electrophoresis is a method for separation and analysis of biomacromolecules ( DNA, RNA, proteins, etc.) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size (IEF ...
and the gel is laid on top of a substrate treated
agar
Agar ( or ), or agar-agar, is a jelly-like substance consisting of polysaccharides obtained from the cell walls of some species of red algae, primarily from ogonori (''Gracilaria'') and "tengusa" (''Gelidiaceae''). As found in nature, agar is ...
ose.
Zymography can also be applied to other types of enzymes, including
xylanase
Endo-1,4-β-xylanase (EC 3.2.1.8, systematic name 4-β-D-xylan xylanohydrolase) is any of a class of enzymes that degrade the linear polysaccharide xylan into xylose, thus breaking down hemicellulose, one of the major components of plant cell w ...
s, lipases and chitinases.
See also
*
SDS-PAGE
SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular masses between 5 and 250 kDa. T ...
References
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Laboratory techniques
Molecular biology