Recent Patents on Biotechnology 2009, 3, 175-184

175

Protease Analysis by Zymography: A Review on Techniques and Patents

Jeff Wilkesman1* and Liliana Kurz2
1

Universidad de Carabobo, Facultad de Ciencias y Tecnologa, Departamento de Qumica. Av. S. Allende, Brbula,
2005, Valencia, Venezuela; 2Universidad de Carabobo, Facultad de Ingeniera, Escuela de Ingeniera Qumica.
Av. Universidad, Brbula, 2005, Valencia, Venezuela
Received: June 15, 2009; Accepted: June 29, 2009; Revised: July 16, 2009

Abstract: Zymography, the detection of enzymatic activity on gel electrophoresis, has been a technique described in the
literature for at least in the past 50 years. Although a diverse amount of enzymes, especially proteases, have been detected,
advances and improvements have been slower in comparison with other molecular biology, biotechnology and
chromatography techniques. Most of the reviews and patents published focus on the technique as an element for
enzymatic testing, but detailed analytical studies are scarce. Patents referring to zymography per se are few and the
technique itself is hardly an important issue in titles or keywords in many scientific publications. This review covers a
small condensation of the works published so far dealing with the identification of proteolytic enzymes in electrophoretic
gel supports and its variations like 2-D zymography, real-time zymography, and in-situ zymography. Moreover, a scope
will be given to visualize the new tendencies of this method, regarding substrates used and activity visualization. What to
expect from zymography in the near future is also approached.

Keywords: Electrophoresis, enzymes, in-situ-zymography, matrix metallo-proteases, multiple-layer-zymography, proteases,

two-dimensional zymography.
INTRODUCTION
Zymography is known as an electrophoretic technique,
commonly based on sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), which contains a
substrate copolymerized within the polyacrylamide gel
matrix, for the detection of an enzymatic activity [1].
Samples are normally prepared by the standard SDS-PAGE
treatment buffer, under non-reducing conditions, i.e. absence
of heating and reducing agent [2-mercaptoetanol,
dithiothreitol (DTT)]. After the electrophoretic run, the SDS
is soaked out from the gel (zymogram) by incubation in a
non-buffered Triton X-100 (or similar detergent), followed
by incubation in an appropriate activation buffer, for an
optimized length of time and temperature, depending on the
type of enzyme being assayed and the type of substrate being
degraded. The zymogram is subsequently stained, and areas
of digestion are distinguished.

enzyme inhibitor activity. Following staining, areas of

inhibition are visualized as dark bands against a clear (or
lightly stained) background [1,4].
Some of the most important articles will be mentioned in
the coming sections. First, we will focus on the evolution
that zymography have had, and the importance of proteases
and its analysis by zymography. Afterwards, a view over
patents and proteases will be given. Then, a small scope
concerning the analytics behind zymography and how to
quantify enzymatic activity will be discussed. Finally,
different types of zymography will be described Fig. (1),
including one-dimensional (1-D), two dimensional (2-D),
zymography, real time zymography (RTZ) and in-situ
zymography (ISZ).
ZYMOGRAPHY EVOLUTION

Though many different types of zymography exist

(according to the type of enzyme), not all are possible to
mention in this paper. Specialized literature is available for
protocols of many enzymes, i.e. there are more than 900
different methods reported for the detection of more than 400
different enzymes [2]. For the specific case of proteases,
gelatin is one of the most frequently used substrate. In this
case, visualization of the proteolytic activity appears as clear
bands over a deep blue background, after Coomassie staining
[2,3]. Another technique, reverse zymography, copolymerizes both the substrate and the enzyme within the
acrylamide matrix, and is useful for the demonstration of

Zymogen, from the Greek zymo (ferment, leaven) and

gen (new, beginning), is a term used to define an inactive
enzyme precursor (or proenzyme). The term zymogram
refers to a record of the enzymatic activity. In 1957, the first
article that demonstrated the separation and visualization of
esterase activity from crude tissue extracts by zone
electrophoresis in starch gels, using histochemical staining
methods, was published [5]. The resulting electropherograms
with the demonstrated enzymatic activity was called
zymograms. A year later, another article was published
concerning the histochemical characterization of hydrolases
[6]. In the following years, several more articles were
published concerning interesting features about the new
zymographic technique [7-12].

*Address correspondence to this author at the Universidad de Carabobo,

Facultad de Ciencias y Tecnologa, Departamento de Qumica. Av. Salvador
Allende, Brbula, 2005, Valencia, Venezuela. Tel: +58 241 868 82 29;
Fax: +58 241 868 82 29; E-mail: jwilkesm@uc.edu.ve

In the decade of the 60s, over 200 articles related with

zymography were published. As reviews, two broad scope
articles were written concerning zymography as a relevant

1872-2083/09 $100.00+.00

2009 Bentham Science Publishers Ltd.

176 Recent Patents on Biotechnology 2009, Vol. 3, No. 3

Wilkesman and Kurz

Fig. (1). Schematic overview of some zymographic techniques. (A) 1-D zymography consists in a SDS-PAGE, with a co-polymerized
substrate. After run and enzyme activation, active bands corresponding to the enzymes are seen as white bands with a blue background. (B)
For the 2-D zymogram, the sample must be first submitted to IEF and then to SDS-PAGE with the co-polymerized substrate. After run and
incubation, white spots are evidence of enzymatic activity. (C) The spots obtained by 2-DZ, may be further analyzed by MALDI-TOF/MS,
in order to identify the enzyme. (D) In-situ zymography (ISZ) is performed directly on tissue samples, allowing cellular localization of the
enzymatic activity. (E) Real-time zymography (RTZ) allows continuous detection of the enzymatic activity over time. (F) Multiple layer
substrate zymography (MLSZ) allows the simultaneous detection of different kind of enzymes from one gel. A gel (1) is run with the sample
and then further electrotransferred to several zymograms (2, 3 and 4) containing different substrates. The net result is that with one run, it is
possible to detect different enzymes. (G) Reverse zymography consists in incorporating not only a substrate to the gel, but also the enzyme.
Thus, it is possible to detect enzyme inhibitors, visible as dark bands on a light background.

technique for several enzyme characterizations and posterior

cytogenetical, embryological and ecological studies [13,14].
In the 70s, over 500 articles with close relationships
with zymography were published. Relevant in this decade is
the only review found, concerning the study of human serum
cholinesterase [15]. In the 80s over 800 articles appeared
with zymogram as keyword. Reviewed were enzymes like
acid phosphatase and esterase, involving the zymogram
technique [16,17]. An interesting article by Heussen and
Dowdle, describes the technique of zymography using SDSPAG with copolymerized gelatin [18]. In 1982, a method for
making zymograms to test serum leucine aminopeptidase
was patented [19].
In the 90s almost 3000 articles concerning zymograms
were published, 3 of them review articles, with important
enzymes notes [20-22]. A remarkable publication is the one
from Lantz and Ciborowski [1], where they carefully
analyzed many of the variables affecting the enzyme activity
during the electrophoretic separation and posterior enzyme
activation process. They identified and characterized
microbial proteases using SDS-PAGE and PAGE in nondissociating gels. Protease separation by SDS-PAGE is

limited by the fact that some enzymes do not renature and

hence cannot be detected. If, however, the protease is SDSresistant, its relative molecular weight (MW) can be
estimated. Protein separation using non-dissociating PAGE
allows protease to be active in the absence of SDS,
permitting detection of multiple forms of enzymes; yet it
cannot be used to estimate its MW. An important variable to
control during development of zymograms was the length of
time of incubations. Increasing incubation times usually
increases the sensitivity of protease detection, but also
increases the diffusion of the proteases and substrates.
Moreover, clear zones of hydrolysis produced by closely
migrating enzymes will join, making impossible the
detection of all proteolytic species in a sample [1].
Among the advantages of zymography are that it permits
the assessment of a repertoire of enzymes that have a
particular activity in crude cell extracts; it gives an
estimation of the MW and pI of the enzyme(s); and it allows
to identify and monitor specific and non-specific enzymatic
activities in complex biological and clinical samples [23,24].
In general, the combination of versatile enzyme assay
techniques with SDS-PAGE offers a powerful means for

Advances in Zymography Techniques and Patents Concerning Protease Analysis

meeting the increasing demand for the high-throughput

screening arising from protein engineering, combinatorial
chemistry, and functional genomics, as recognized by
Bischoff et al. [25].
Beginning 2000 and until June 2009, there has been a
total of over 8000 scientific publications related with the use
of zymograms. There is an apparent exponential tendency in
the increase of the number of publications through the
decades. If this tendency keeps its pace, we would be
expecting over 16,000 publications related with zymography
by the end of 2020 Fig. (2). This illustrates the impact that
zymography has had over science life since its beginnings.

Recent Patents on Biotechnology 2009, Vol. 3, No. 3

177

According to the enzyme nomenclature of the International Union of Biochemistry and Molecular Biology [28],
hydrolases belong to class 3, and peptidases to the subclass
3.4. This subclass can be divided depending on the type of
reaction catalyzed into: a) exopeptidases or peptidases,
which are proteases catalyzing the splitting of peptide bonds
at either the N- or C- terminus of the substrate; and b)
endopeptidases or proteinases, which are proteases splitting
the peptide bonds within the protein substrate. Endopeptidases act preferentially in the inner regions of the peptide
chain and are further classified according to their active site
into: serine, cysteine, aspartic and metallo-proteases.
Serine Proteases
This is the most studied class of enzymes, which is
characterized by a serine residue at its active site. There are
two different families: the mammalian serine proteases and
the bacterial serine proteases [29,30]. Subtilisin is the most
representative example from the latter family. On the other
hand, representative enzymes belonging to the mammalian
family are trypsin, chymotrypsin, elastase and kallikrein. It is
believed that these enzymes evolved from a common
ancestor. They are assumed to have acquired different specificities by mutation of the genes descended from an
evolutionary precursor [30].
Cysteine Proteases

Fig. (2). Exponential tendency observed for the number of

publications appeared on peer-reviewed journals since 1960. A total
of 12794 publications appear so far. This number should be slightly
higher by the end of 2009, fitting the tendency better. Data was
obtained using the Highwire Press search machine from Stanford
University (http://highwire.stanford.edu/, date of access July/11/
2009), with the keyword zymogra*. All databases (including
PubMed) were selected.

Formerly known as thiol proteases, this group of enzymes is characterized by a thiol group of a cysteine residue at
its active site. Cysteine proteases have been isolated from
plants, animals and bacteria. The most representative
enzymes are papain, actinidin, stem bromelain, ficin, cathepsins B and H, streptococcal proteinases, clostripain, and the
cytosolic calcium activated proteases (calpains). There has
been an increasing interest in these proteases, especially for
modification of food proteins and for synthesis of biologically active peptides and their analogues [31].

PROTEASES AS MAIN TARGET ENZYMES

Aspartic Proteases

Proteases, also known as proteinases, proteolytic enzymes or peptidases, are enzymes that hydrolyze the peptide
bond of proteins; hence, they are all hydrolases. Proteases
are normally generated as an inactive proenzyme (zymogene), and according to requirements, converted into the
active form through limited proteolysis. Proteolytic enzymes
are ubiquitous, being distributed in all biological fluids and
tissues [26].

Due to their optimal activity at pH 1.5-5, the aspartic

proteases were first called acid proteases. After the
identification of particular carboxyl groups, essential for
catalysis, they were called carboxyl proteases. Later, it was
found that all enzymes of this type have an aspartyl residue
at their active site, giving the actual and more appropriate
name of aspartic proteases, since some enzymes belonging to
this group work optimally at neutral pH [32]. These enzymes
cleave preferably bonds containing Phe, Met, Leu, or Trp,
and the catalytic activity has a pH optimum of about 2.0.
Pepsin is one of the most relevant enzymes belonging to this
group. Other representative enzymes are renin (vertebrate
kidney enzyme), cathepsin D, penicillopepsin (from
Penicillium janthinellum) and chymosin [32].

In general, proteases accomplish two major functions: (i)

a regulatory function, which involves activation or
inactivation of specific proteins by selective proteolysis, and
(ii) a general proteolytic function, which is a less specific
process, resulting in the bulk breakdown of cellular proteins.
These degradative mechanisms remove denatured proteins as
well as facilitate adaptive responses by destroying native
proteins no longer needed by the cell. Both types of proteolysis are highly regulated and usually occur in response to
specific extracellular signals, such as gametogenesis, differentiation, and tissue development [27].

Metalloproteases
The metalloproteases are characterized by bearing a
metal ion at the active site, usually Zn, although in some
instances, other transition metals can substitute it. Two
families are known: the mammalian pancreatic carboxypep-

178 Recent Patents on Biotechnology 2009, Vol. 3, No. 3

tidase A, and the bacterial thermolysin. Both of them are Zn

metallo-enzymes, and have similar active site configurations.
These enzymes do not form covalent enzyme complexes
(like the aspartic proteases) [33]. The matrix metalloproteinases (MMPs) are a major group of metalloproteases,
which regulate cell matrix composition and are known for
their ability to cleave one or several extra-cellular matrix
constituents, as well as non-matrix components. The role of
MMPs has been identified in normal and pathological
processes as embryogenesis, wound healing, inflammation,
arthritis and cancer [34,35]. MMPs involved in extracellular
matrix degradation are subject to regulatory controls at
multiple levels including transcription, mRNA stability,
translation, secretion, activation of proenzymes, and degradation and inhibition by specific endogenous inhibitors
known as tissue inhibitors of metalloproteinases (TIMPS)
[35].
Proteases are key regulators of a wide range of biological
processes in all living organisms, given their highly specific
hydrolysis of peptide bonds. This proteolytic processing
regulates the activity and the compartmentalization of many
proteins and consequently of many cellular processes.
Proteases constitute an attractive potential as therapeutic
targets, for dysfunction in the protease expression and its
activity is involved in various pathological conditions, e.g.
cardiovascular and neurodegenerative diseases, arthritic
diseases, infection, and cancer [24]. Zymography has been
used to study extracellular matrix (ECM)-degrading enzymes, in particular the MMPs [36].
PROTEASES, ZYMOGRAPHY AND PATENTS
Though many articles have been published concerning
the zymography technique, not so many patents appear in
this respect. A zymography kit for the gel preparation has
been patented, where a novel substrate protein is used,
without limiting the use of gelatin. The novel protein is
incorporated into the gel by a cross-linking agent [37].
Patents concerning zymography and MMPs are the most
abundant, e.g. a method for estimating the efficiency of
arterial hypertension therapy, by determining the content of
MMP-9 using zymography, was patented [38]. Another
related patent is a kit for diagnosing pregnant state and a
method capable of reconsidering assisted reproduction
technology, based on the activity of MMP-9. The activity of
MMP-9, present in the follicular fluid retrieved from women
who attended an in vitro fertilization program, is measured
by zymography and pregnancy is evaluated by measuring the
activity of MMP-9. According to the patent, the chance of
pregnancy is zero when a low MMP-9 activity is registered;
whereas a high activity increases pregnancy chances up to
60%. Thus, prediction of pregnancy in assisted reproduction
technology measuring MMP-9 activity is possible [39].
A non-invasive method for facilitating the diagnosis of
cancer with epithelial origin has been patented. Detection of
matrix metalloproteinase (as cancer indicator) in urine
samples were performed by zymography [40]. Another
method for determining the susceptibility to a chronic
obstructive pulmonary disorder (COPD) that comprises the
determination of the presence of an exon 6, codon 279
Gln/Arg single nucleotide polymorphism within the MMP-9

Wilkesman and Kurz

locus in a biological sample (where the 279 Arg polymorphism indicates susceptibility to COPD), has been
patented as well [41].
A method to diagnose the presence of metastatic cancer
and to prognosticate its appearance, by identifying high MW
enzyme complexes comprising MMPs, has been patented
[42]. Equally, an ex-vivo method for diagnosis of proliferative diabetic retinopathy by the determination of MMP-2
and/or MMP-9 gelatinase activity by zymography has also
been patented [43].
Other zymographic methods related to the analysis of
MMPs have been patented as well [44-46]. Besides MMPs,
other recent patent describes a method of obtaining proteases
(serine protease like SplA or SplB) from Staphylococcus
aureus, and their use in the specific hydrolysis of a
polypeptide chain, the amino acid sequence recognized by
them and their use [47].
ANALYTICAL ZYMOGRAPHY
Quantitation of proteolytic activity of samples subjected
to zymography is feasible. Many papers have been published
concerning the analytical and statistical implications of the
method. Worth mentioning is the work of Harcum and
Bentley, where intracellular proteases from recombinant E.
coli were quantified [48]. It has been reported that up to
picograms of gelatinase may be analytically quantified [49].
An enhanced method was further developed using a singlestep staining-destaining procedure, leading to faster and
more reproducible results during quantification [50].
Subpicograms of collagenase have been possible to
detect with zymography. The use of casting native collagen
type I in SDS-PAGE for the determination of interstitial
collagenase has been described. The addition of collagen in
the gels reduces protein migration, making mandatory the
corrections for an accurate MW evaluation. The method was
extremely sensitive, being possible to detect 0.1 pg of (4aminophenyl) mercuric acetate (APMA)-activated procollagenase. A patent request was registered in 1997, but the
proper document was not found [51].
The extent of gelatin degradation has been measured with
an EDC scanning densitometer, being the recorded value
directly proportional to the amount of enzyme. This method
of gelatinase activity determination was quantified from the
gel by assaying hydroxyproline as an index of gelatin
breakdown [52].
The comparison of gelatin, casein and fibrin as substrates
for zymography has also been studied and its effect over
enzyme activity and over electrophoresis studied [53]. The
application of this quantitative technique in analyses of
MMP-2 and MMP-9 related with breast cancer has been
published recently [54].
1-D AND 2-D ZYMOGRAPHY
The standard method for 1-D zymography is based on the
use of SDS-PAGE co-polymerized with a protein substrate
(Fig. (3), steps 1-3). Normally gelatin, casein, or fibrin is
used. Proteases that have the ability to renature after removal
of SDS and to exert proteolytic activity on a co-polymerized

Advances in Zymography Techniques and Patents Concerning Protease Analysis

Recent Patents on Biotechnology 2009, Vol. 3, No. 3

179

substrate can be analyzed with this method. E.g., MMP-2

(gelatinase A, 72 kDa) and MMP-9 (gelatinase B, 92 kDa)
can be detected on gelatin zymograms and MMP-7 on casein
gels. Coomassie Blue staining of the gel reveals sites of
proteolysis as translucent bands on a dark blue background
[24,54]. Other proteases may be used as molecular weight
standards, e.g. collagenase type I (140 kDa), thermolysin (37
kDa), chymotrypsin (30 kDa) and trypsin (19 kDa) [55].
Some of the advantages that zymography posses are the
use of inexpensive materials, relatively short assaying times,
and proteases with different MWs, showing activity towards
the same substrate, can be detected and quantified on a single
gel. Some proteases, like the MMPs, are often associated
with TIMPs when in solution, but during electrophoresis, the
inhibitors dissociate from the MMP, permitting the detection
of the enzymatic activity. E.g. gelatin zymography is a
common method to study MMP-2. However, it has been
reported that the method is insufficient to provide
meaningful information about the status of MMP-2 and
parallel methods must be performed in order to check in vivo
conditions [56]. Some recently published patents make use
of zymographic techniques [47,57,58].
Typical substrates for zymography are gelatin, casein and
fibrin, allowing also the detection of proteolytic activity in
cell or tissue homogenates. The MW of the proteolytic bands
can be determined by using appropriate MW standards [55].
The type of protease can be established by comparison with
recombinant proteins and the use of specific protease
inhibitors. Based solely on zymogram techniques, nevertheless, no information on the localization of the proteolytic
activity in cells or tissues can be obtained [24].
A method for protease detection in a sample, by
incubating the sample with a substrate and observing
proteolytic cleavage of the substrate has been patented [59].
The substrate is a modified proenzyme containing a recognition site, i.e. an activation site, cleavable by the protease.
The protease may be an aspartic protease or a metalloprotease, and the modified proenzyme may be pro-urokinase,
having a mutant activation site, which is cleavable by the
protease to be assayed [59].
Since 1997, a new approach on zymography was
achieved, once the IEF was incorporated as a first step of
sample separation, followed then by classical zymography
(Fig. (3), steps 4-6). This gave name to the two-dimensional
(2-D) zymography. 2-D zymography combines 2-D electrophoresis with zymography, and was used to analyze
proteases and other proteins produced by different phase
variants of two strains of Photorhabdus luminescens [60]. In
1998, another work used the 2-D zymography to detect
proteolytic enzymes in human pure pancreatic juice (PPJ).
The authors found that their results suggested that the spots
of MW 70 kDa and pI 5.3-5.5 in PPJ of pancreatic cancer
might be MMP-2 [61].
If the zymogram is run in parallel with a normal SDSPAGE, after staining both gels, they may be superimposed
and compared Fig. (3). After analysis, spot patterns may be
related. With the use of matrix-assisted laser desorption
ionization time of flight (MALDI-TOF) mass spectrometry,

Fig. (3). Schematic overview of 1-D and 2-D zymography. (1) The
sample, a crude extract or up to picograms of a purified active
enzyme, is diluted in sample buffer under denaturing (SDS) but
non-reducing conditions (absence of 2-mercaptoethanol, DTT, and
heat) in order to submit it to SDS-PAGE or zymography. (2)
Electrophoresis of the sample gives the band pattern and MW
estimates of the total protein content. (3) The zymogram is an SDSPAG with a proper substrate copolymerized (gelatin, casein, fibrin),
allowing visualization of the bands corresponding only to the active
enzyme. Correlation of the band(s) with the electrophoresis is
normally done, as the copolymerized substrate might exert an effect
on protein migration. (4) The sample is treated and submitted to
IEF in order to separate the proteins according to their pI. This
results in the first dimension of the separation. (5) The IPG-Dalt
strip used for IEF is now loaded over a SDS-PAG to run the second
dimension, separating according to MW. (6) A parallel strip is
loaded over the zymogram. After run, gel is incubated in proper
activation buffer and stained. White spots indicate the presence of
enzymatic activity. MW and pI of the enzyme(s) may now be
determined.

the spots may be further analyzed and identified, a field that

some researchers has recently named zymoproteomics Fig.
(1C). This combined method of 2-D gel and zymo-graphy is
a powerful tool to detect serine proteases, as published by
Park et al. [62]. An enhanced method was developed shortly
after, in which SYPRO Ruby dye (a sensitive fluorescencebased method for detecting proteins) was used [63].
Further investigation stated that mild denaturing conditions required for IEF in the first dimension may alter the
interpretation of the 2-D zymography, being necessary to
take care during sample preparation [64]. Another work
describes a zymographic method for the detection of pro-

180 Recent Patents on Biotechnology 2009, Vol. 3, No. 3

teases using quenched fluorescent substrates. The enzymes

were separated using 1-D and 2-D electrophoresis, and the
gel was then incubated with the quenched fluorescent
substrate. Afterwards, using UV light, the proteases were
directly localized by the released fluorescence. The protease
spots were cut from the gel and processed for mass
spectrometry (MS) identification [65].
As can be seen, 2-D zymography can be used to develop
or test substrate specificity and to detect the proteases that
are able to cleave a given substrate in a complex biological
fluid. An advantage of the technique is that direct identification of proteases is possible without complex purification.
REAL TIME ZYMOGRAPHY
Another methodology proposed is the 1-D and 2-D realtime zymography (RTZ) and real-time reverse zymography
(RTRZ) [66]. A fluorescein-isothiocyanate-labeled substrate
had been used to develop zymographic and reverse
zymographic methods to detect MMPs and TIMPs. With the
use of a transilluminator, the zymograms can be visually
monitored without stopping the enzymatic reaction, thus
being called real-time zymography Fig. (1E) and realtime reverse zymography. The advantages are that, because
the reaction can be constantly monitored on the
polyacrylamide gels, (i) the incubation time can be easily
optimized; (ii) a higher sensitivity is achieved with lower
substrate amounts; (iii) a semi-quantitative analysis of MMP
is possible; and (iv), in the case of the RTRZ, the inhibitor
bands can be easily distinguished from other proteins,
because the fluorescence detection is specific for substrate
digestion [67].
A method for detecting and assaying enzymatic activity
and its inhibitor by RTZ has been patented [68]. The method
provides a way to detect proteolytic activity and the effect of
a proteinaceuos inhibitor, with higher sensitivity and better
quantitation than conventional zymography. The method
involves a fluorescence-labeled substrate in the gel.
SPECIFIC DETECTION OF ENZYMES
Zymography bares, nevertheless, a number of
disadvantages (according to the enzyme being analyzed), i.e.
special substrate requirements, need of co-factors, and
difficulties in distinguishing enzymes with overlapping
activities. In some cases, these drawbacks have slowed the
use of zymography as a routine laboratory method. Yet, it
has been published [23], that difficulties are overcome by
employing small-defined substrates, covalently attached to
the gel matrix. Furthermore, the assay is compatible with 2-

Wilkesman and Kurz

D zymography, thus having a great potential in the highthroughput screening and characterization of complex
biological and clinical samples. A synthetic ribonucleotide
(ACAGUAUUUG) was linked at the 3 end via an 18-link
spacer to an acrylamide group Fig. (4), which was incorporated using Acrydite phosphoramidite. This acrylamideoligonucleotide was radiolabeled at the 5 end, co-polymerized into standard SDS-PAG and used for the in-gel
activity staining of several enzymes (mammalian RNase A,
bacterial alkaline phosphatase, and T4 polynucleotide
kinase) [23].
Another specific method patented uses fluorescencequenched substrates to assay proteolytic enzymes [69]. The
method consists in incubating an enzyme-containing sample
with an immobilized fluorescence-quenched peptide (QueSub-Flu-Spa-Car or Flu-Sub-Que-Spa-Car), where Que is a
quencher, capable of absorbing fluorescent radiation emitted
by the fluorophore, Sub is a peptide chain that contains a
specific cleavage site for the protease; Flu is a fluorophore;
Spa is a direct bond or a spacing chain; and Car is a waterinsoluble and/or macromolecular carrier. Afterwards, the
carrier material is irradiated and the fluorescence is
measured [70].
MEMBRANE PROTEINS ANALYSIS
Another issue concerning 2-DZ is the analysis of
membrane proteins. Conventional 2-DE technique is
inconvenient for membrane protein analysis, basically due to
its high hidrophobicity, low capacity of the 2-DE lysis buffer
to extract membrane proteins from the lipid environment and
keep them solubilized, and tendency to aggregation during
IEF. A recent publication [71] recommends the substitution
of the IEF as first dimension with electrophoresis using
cationic detergents, explicitly 16-benzyldimethyl-n-hexadecylammonium chloride (16-BAC) and cetyl trimethyl
ammonium bromide (CTAB), or the anionic detergent SDS.
The separation of native membrane protein complexes
through the application of blue and clear native gel electrophoresis (BN/CN-PAGE) and the free-flow electrophoresis
(FFE) of membranes are reviewed in the article [71].
Another article [72] dealing with membrane proteomics
proposes an alternative technique, 2-D BAC/SDS-PAGE (2DB) utilizing the cationic detergent benzyldimethyl-nhexadecylammonium chloride (BAC) in the first dimension
and SDS in the second dimension. 2-DB permitted the
identification of highly hydrophobic proteins. The application of tube gels in the first dimension, and the introduction of improved buffer systems represent a huge
potential for future 2-DB-based membrane studies [72].

Fig. (4). Chemical structure of the Acrydite-modified oligonucleotide used as a small defined substrate in zymography (according to [23]).

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Recent Patents on Biotechnology 2009, Vol. 3, No. 3

181

IN-SITU ZYMOGRAPHY
In-situ zymography (ISZ) was introduced some decades
ago to localize MMP activity. The procedure, based on
zymography using SDS-PAG with co-polymerized gelatin,
casein, or fibrin, brings in contact a tissue section or cell
preparation employing a photographic emulsion containing
gelatin or a fluorescence-labeled protein substrate. After
incubation, enzymatic activity is revealed as white spots in a
dark background or as black spots in a fluorescent background Fig. (5). Due to limited sensitivity, nevertheless, this
approach hinders exact localization of proteinase activity
[24,73,74]. A patent from 2002 explains alternative dyeing
methods to measure protease activity through ISZ [75].

Fig. (6). Molecular structure of FITC used in ISZ.

though, that ISZ has limitations regarding quantitation of

protease activity and consequently cannot replace gel
zymography [24].
ISZ has been used to check the inhibitory effect of
MMPs, and its methodology has been patented [76]. E.g., a
gelatinolytic activity of MMPs was determined by ISZ, using
FITC-DQ gelatin intramolecularly quenched as substrate.
This method was used to confirm the inhibitory activity of a
monoclonal antibody [anti cobalttetracarboxylated phenylporphyrin (CoTCPP mAb)] at cellular level. The effect of the
antibody was examined on gelatinolytic activity of human
fibrosarcoma HT1080 cells that constitutively secrete MMP2 and 9 [77]. As demonstrated through fluorescent micrographs of ISZ, the untreated human fibrosarcoma HT 1080
cells exhibited significant cell surface gelatinase activity.
However, in the presence of 1
M anti CoTCPP mAb,
gelatinase activity was reduced as compared to that observed
in control cells. These results demonstrated that anti
CoTCPP mAb inhibited MMP-2 and MMP-9 at the cellular
level [77].

Fig. (5). Schematic overview of in-situ zymography. The method

may be performed using (A) a photographic emulsion, or (B) a
fluorescent substrate. (A) 1. A tissue section is laid over the slide. 2.
The slide is cover with photographic emulsion. 3. Slide is
incubated. 4. After development and fixation, protease activity is
visaulized as white spots over a dark background. (B) 1. An empty
slide is coated with a fluorescent labeled substrate. 2. Substrate is
uniformly extended over the slide and excess is removed. 3. A
tissue section is applied over the slide. 4. After incubation, protease
activity is visualized as dark spots over a fluorescent background.
Alternatively, tissues may be previously treated with specific
inhibitors and then ISZ is performed. A reduction of the white spots
(A) or dark spots (B) indicates enzymatic inhibiton (According to
[4, 74]).

Sensitivity was improved when dye-quenched (DQ)gelatin was introduced. This DQ-gelatin is heavily labeled
with fluorescein isothiocyanate (FITC) Fig. (6), so that its
fluorescence is quenched. Cleavage of DQ-gelatin by
proteases produces fluorescent peptides, visible against a
weakly fluorescent background. The role of specific proteinases in various physiological and pathological conditions
has been better understood with ISZ, especially when used in
parallel with other techniques like immunohistochemistry,
in-situ hybridization and Western blotting. It must be stated

Another technique recently developed is film in-situ

zymography (FISZ). Regional detection of tissue MMP
activities has been possible employing a polyester film
coated with gelatin uniformly and thinly. After incubation,
the films were stained with Biebrich Scarlet, and the
unstained area corresponded to MMP activities. This kind of
detection by FISZ has resulted very simple and quantitative,
and constitutes a handy tool for the analysis of many diseaseinvolved MMPs [78].
Some protocols concerning ISZ have been patented, e.g.
a mammal that received a compound exhibiting MMPinhibitory activity has been tested [57,79]. A thin mem-brane
protocol for measuring MMP-7 has also been patented [80].
APPLICATION AREAS OF ZYMOGRAPHY AND
PATENTING IMPACT
The analysis of enzyme activity by electrophoresis
provides useful information in biochemistry and biology.
Isozymes and allozymes, used as gene markers, enable
advances in enzymology, molecular evolution and genetics.
They also serve to solve problems in systematics and
phylogenetic relationships among species. Many fields have
benefit with the advances of zymography, including clinical
and diagnostic medicine, medical genetics, agricultural
entomology, genetic monitoring of environmental pollution,
forensic science, etc. [2].

182 Recent Patents on Biotechnology 2009, Vol. 3, No. 3

Biotechnologically, zymography is still a simple and

powerful tool for the separation and identification of the
second-level structural gene products. For proteomic
purposes, zymography offers a series of advantages when
studying enzymes. These include: a) enzymes and their
structural isoforms are detected and identified; b) isozyme
functional properties may be examined in-gel; c) the genetic
basis of isozymes from individual variation and tissue
specificity may be inferred; d) different enzymes with
similar and overlapping substrate specificities can be
discriminated; e) MW of the enzyme(s) can be determined
by combining native and denaturing condition; f) enzymes
pI is determined by using IEF; g) quaternary structure of the
enzymes(s) may be inferred, as its subunits can be
determined [2]. Given the profound impact that zymography
exerts, it is clear to realize the importance to establish new
and enhanced methodologies for enzymatic analysis, and to
further patent them. Patents concerning clinical testing have
been so far the most abundant ones and perhaps the most
profitable too. And though the most amounts of patents so
far belong to the biomedical field, other fields of impact (e.g.
environment, agriculture) shall not be left unseen in the near
future. According to Fig. (2), we do expect a huge increase
in publications concerning zymography, hence an equivalent
increase is also expected for patents in this topic.
CURRENT & FUTURE DEVELOPMENTS
Actual tendencies in zymography are directed to the
simultaneous detection and identification of several enzymes
on a same gel. A system to detect 3 different hydrolases
(cellulase, lipase, and protease) using a single SDS-PAG and
an electrotransfer system has been developed [81]. Briefly,
after electrophoresis, enzymes in the gel are electrotransferred to three sandwiched substrate gels containing the
different substrates (glycerol tributyrate, azo-carboxymethyl
cellulose (Azo-CMC), and fibrin for the respective detection
of cellulase, lipase, and protease). This method has been
called a multiple-layer substrate zymography (MLSZ, Fig.
(1F)).
The prognosis is that further research will be done in this
respect, surely leading to new nuances of zymography.
Beyond multi-layer zymography, efforts have been done to
achieve poly-substrate gels in order to detect activity of
different enzymes in the same gel [82].
New investigations show the versatility of zymography
for the screening of proteases (collagenases) and amylases in
the analysis of microbial and biochemical properties of hightemperature compost and thermophilic bacteria [83,84].
A recent published work [85] reveals a new scope that
has not yet been applied for zymography. Most proteases
tested so far have resulted to be kinetically stable proteins
(KSPs). Kinetic stability (KS) is a feature used by nature to
allow proteins to maintain activity under harsh conditions
and to preserve the structure of proteins that are prone to
misfolding [85]. The biological and pathological meaning of
KS still remains scarcely understood. It has been proposed
the application of a diagonal 2-D (D2D) SDS-PAGE assay to
identify KSPs in complex mixtures. This assay should allow
the widespread investigation of KS, including the
proteomics-level identification of proteases in different

Wilkesman and Kurz

systems, leading to a better understanding of the biological

and pathological significance of this property of proteins. We
are sure that it will not take long until we meet publications
concerning D2D zymography.
Another yet not so widespread technique is capillary
electrophoresis zymography (CEZ). It has been published
that beta-glucosidases activity in environmental samples can
be analyzed using substrate-incorporated CEZ, and kinetic
parameters could be determined by repeated injections at
different substrate concentrations [86].
With no doubts, 2-D zymography combined with enzyme
identification by MS should constitute the state-of-the-art
technique for further protease research, what can be called
zymoproteomics. Hand in hand with 2-DE, and given
promising complementary technologies (e.g. multidimensional protein identification technology, stable isotope
labeling, protein or antibody arrays), 2-D zymography
represents a method that can be routinely employed for
parallel quantitative expression profiling of large sets of
complex proteases mixtures such as whole cell lysates. 2-D
zymography enables the separation of complex mixtures of
proteins according to pI, molecular mass, solubility, and
relative abundance [87]. The current 2-D zymography
technology employing IPGs, overcomes former restrictions
of carrier ampholyte based 2-DE concerning handling,
resolution, reproducibility and separation of very acidic
and/or basic proteins. The development of IPGs between pH
2.5-12 had enabled the analysis of, e.g., very acidic proteases
like the aspartic protease group. Narrower-overlapping IPGs
have provided an increase in resolution (up to
pH = 0.001)
and, combined with pre-fractionation methods, the detection
of low abundance proteases. According to gel size, % and
pH gradient used, 2-D zymography may resolve more than
5000 enzymes simultaneously and detect and quantify less
than 1 pg of protein per spot [51,87]. Restrictions of
substrate for zymography development should no longer be a
limitation given the possibility to anchor specific synthetic
substrates to the gel matrix.
In the upcoming time, the main impact of zymography
will still be in the medical, clinical, biological, genetic and
toxicological field. 2-DE and 2-DZ can run as far as 20 2-D
gels at a time with thousands of proteins/enzymes per gel
[88]. Post-translational modifications in proteins may be
easily identified in 2-DE gels, since they appear as
distinctive spot clusters, which can be further identified by
MS. Still, difficulties around the analysis of low-abundance
enzymes and integral membrane proteins must be overcome.
In conclusion, 2-D gels will surely remain as a standard
analysis within the near future, being zymography a
preferred technique to be chosen for precise enzyme analysis
in gel electrophoresis.
ABBREVIATIONS
1-D

= One dimensional

2-D

= Two dimensional

2-DE

= Two dimensional electrophoresis

2-DZ

= Two dimensional zymography

APMA

= (4-Aminophenyl) mercuric acetate

Advances in Zymography Techniques and Patents Concerning Protease Analysis

COPD

= Chronic obstructive pulmonary disorder

DTT

= Dithiothreitol

DQ

= Dye-quenched

ECM

= Extracellular matrix

FITC

= Fluorescein isothiocyanate

FISZ

= Film in-situ zymography

ECM

= Extracellular matrix

IEF

= Isoelectric focusing

IPG

= Immobilized pH gradient

ISZ

= In-situ zymography

MMPs

= Matrix metalloproteinases

MS

= Mass spectrometry

MW

= Molecular weight

pI

= Isoelectric point

RTZ

= Real time zymography

RTRZ

= Real time reverse zymography

SDS-PAG

= Sodium dodecyl sulfate - polyacrylamide

gel

SDS-PAGE = Sodium dodecyl sulfate - polyacrylamide

gel electrophoresis
Spl

= Serine protease like

TIMPS

= Tissue inhibitors of metalloproteinases

ACKNOWLEDGEMENTS
This work was partially funded by the Consejo de
Desarrollo Cientfico y Humanstico de la Universidad de
Carabobo (CDCH-UC-XXXX-2009). The authors thank the
support of the Universidad de Carabobo (FACYT and
Faculty of Engineering).
CONFLICT OF INTEREST

Recent Patents on Biotechnology 2009, Vol. 3, No. 3

[8]

[9]

[10]
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[14]
[15]
[16]
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[19]
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[21]

[22]
[23]

[24]
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The authors declare no conflict of interest.

[26]

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