Recent Patents on Biotechnology 2009, 3, 175-184 175

1872-2083/09 $100.00+.00 2009 Bentham Science Publishers Ltd.

Protease Analysis by Zymography: A Review on Techniques and Patents

Jeff Wilkesman1* and Liliana Kurz

2

1Universidad 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 - polyacryla-mide 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.

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, copoly-merizes both the substrate and the enzyme within the acrylamide matrix, and is useful for the demonstration of

*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

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

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].

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

176 Recent Patents on Biotechnology 2009, Vol. 3, No. 3 Wilkesman and Kurz

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 SDS-PAG 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 non-dissociating 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 SDS-resistant, 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

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.

Advances in Zymography Techniques and Patents Concerning Protease Analysis Recent Patents on Biotechnology 2009, Vol. 3, No. 3 177

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.

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.

PROTEASES AS MAIN TARGET ENZYMES

Proteases, also known as proteinases, proteolytic enzy-mes or peptidases, are enzymes that hydrolyze the peptide bond of proteins; hence, they are all hydrolases. Proteases are normally generated as an inactive proenzyme (zymo-gene), 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].

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 proteo-lysis are highly regulated and usually occur in response to specific extracellular signals, such as gametogenesis, diffe-rentiation, and tissue development [27].

According to the enzyme nomenclature of the Inter-national 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. Endopep-tidases 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 speci-ficities by mutation of the genes descended from an evolutionary precursor [30].

Cysteine Proteases

Formerly known as thiol proteases, this group of enzy-mes 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, cathep-sins 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 biolo-gically active peptides and their analogues [31].

Aspartic Proteases

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].

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 Wilkesman and Kurz

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 metallo-proteinases (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 degra-dation 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 enzy-mes, 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

locus in a biological sample (where the 279 Arg poly-morphism 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 proli-ferative 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 single-step 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 (4-aminophenyl) mercuric acetate (APMA)-activated procolla-genase. 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, never-theless, 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 recog-nition site, i.e. an activation site, cleavable by the protease. The protease may be an aspartic protease or a metallo-protease, 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 electro-phoresis 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 SDS-PAGE, 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 SDS-

PAG 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 fluorescence-based method for detecting proteins) was used [63].

Further investigation stated that mild denaturing condi-tions 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 Wilkesman and Kurz

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 identi-fication of proteases is possible without complex purifi-cation.

REAL TIME ZYMOGRAPHY

Another methodology proposed is the 1-D and 2-D real- time 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 real-time 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-

D zymography, thus having a great potential in the high-throughput 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 incor-porated using Acrydite phosphoramidite. This acrylamide-oligonucleotide was radiolabeled at the 5 end, co-poly-merized 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 fluorescence-quenched substrates to assay proteolytic enzymes [69]. The method consists in incubating an enzyme-containing sample with an immobilized fluorescence-quenched peptide (Que-Sub-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 water-insoluble 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-hexade-cylammonium 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 electro-phoresis (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 (2-DB) utilizing the cationic detergent benzyldimethyl-n-hexadecylammonium chloride (BAC) in the first dimension and SDS in the second dimension. 2-DB permitted the identification of highly hydrophobic proteins. The appli-cation of tube gels in the first dimension, and the intro-duction 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]).

Advances in Zymography Techniques and Patents Concerning Protease Analysis 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 back-ground 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. (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 protei-nases 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

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 phenyl-porphyrin (CoTCPP mAb)] at cellular level. The effect of the antibody was examined on gelatinolytic activity of human fibrosarcoma HT1080 cells that constitutively secrete MMP-2 and 9 [77]. As demonstrated through fluorescent micro-graphs 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].

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 disease- involved MMPs [78].

Some protocols concerning ISZ have been patented, e.g. a mammal that received a compound exhibiting MMP-inhibitory 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 Wilkesman and Kurz

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 electrotrans-ferred 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 high-temperature 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

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. multidimen-sional 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 Recent Patents on Biotechnology 2009, Vol. 3, No. 3 183

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

The authors declare no conflict of interest.

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