Zymogram
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Zymography is an
electrophoretic Electrophoresis is the motion of charged dispersed particles or dissolved charged molecules relative to a fluid under the influence of a spatially uniform electric field. As a rule, these are zwitterions with a positive or negative net ch ...
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 hydrolysis ...
enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...
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 () 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 be referred to as hydrolyzed collagen, coll ...
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 through hydrolysis, playing a major role in degradation of extracellular matrix and tissue remodeling. Gelatinases are a type of matrix metalloproteinase (MMP), a family of enzymes that depend on ...
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, 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 and their use in brewing, wine mak ...
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 electrophoresis, discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular m ...
. 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 () 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 be referred to as hydrolyzed collagen, coll ...
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 is the motion of charged dispersed particles or dissolved charged molecules relative to a fluid under the influence of a spatially uniform electric field. As a rule, these are zwitterions with a positive or negative net ch ...
, 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-tetr ...
, 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 finge ...
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 fr ...
), 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 In biochemistry, glycoside hydrolases (also called glycosidases or glycosyl hydrolases) are a class of enzymes which catalyze the hydrolysis of glycosidic bonds in complex sugars. They are extremely common enzymes, with roles in nature inclu ...
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 Enzyme activity, activity. Enzymes are proteins that speed up chemical reactions necessary for life, in which Substrate (biochemistry), substrate molecules are converted ...
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 an electrophoresis method for separation and analysis of biomacromolecules (DNA, RNA, proteins, etc.) and their fragments, based on their size and charge through a gel. It is used in clinical chemistry to separate ...
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" and " tengusa". As found in nature, agar is a mixture of two components, t ...
ose. Zymography can also be applied to other types of enzymes, including
xylanase Endo-1,4-β-xylanase (, 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 walls: : En ...
s, lipases and chitinases.


See also

*
SDS-PAGE SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a Discontinuous electrophoresis, discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular m ...


References

{{reflist Molecular biology techniques