Abstract
Zymography is a commonly used biochemical technique for detecting and analyzing enzyme activity using gel electrophoresis and substrate staining. This review traces the chronological advancements in zymography, from its origins with the pioneering fibrin-plate methods in the 1940s to the recent innovations in multi-substrate applications. It underscores early pivotal developments and acknowledges the overlooked contributors who laid the groundwork for zymography's current methodologies. Current advancements, particularly in gel casting, are highlighted for their ability to facilitate the concurrent analysis of multiple substrates, thereby improving the efficiency, and reducing the environmental impact of proteolytic assays. These methodological enhancements offer a more detailed understanding of enzyme activity and specificity and represent a shift towards more economical laboratory practices. Additionally, the review identifies persisting challenges such as substrate limitations and detection sensitivities, offering insights into potential research directions that could further enhance the technique's capabilities. The review concludes with a discussion on the necessity for standardized sensitivity and detection metrics across various zymographic methods and highlights the utility of zymography in proteomic research, with implications for the development of diagnostic and therapeutic applications. Zymography, as performed today, is a biochemical technique that allows the semi-quantitative detection of proteases and other hydrolases following their electrophoretic separation in gel matrices. Almost every zymography protocol that is used in routine laboratory work or as a starting point in teaching courses, follows from Heussen & Dowdle’s paper from 1980. But a thorough investigation into the origins of this technique takes us eight decades back to the 1940s. In the realm of zymographic techniques, the foundational work of Permin, a graduate student training under Tage Astrup, as well as the subsequent methodological refinements by Astrup & Müllertz in 1952, mark pivotal milestones in the Qeios ID: X0IB4R · https://doi.org/10.32388/X0IB4R 1/15 evolution of the fibrin-plate method for assessing fibrinolytic activity. Permin's initial approach, as detailed in his 1947 publication in Nature, involved the strategic placement of a plasmin-containing solution on a fibrinogen-thrombin matrix in a petri dish, leading to a discernible circular zone of fibrinolysis, with the radius being indicative of the plasmin concentration. This method provided an early, yet profound, quantitative measure of enzyme activity. Building upon this, Astrup & Müllertz's 1952 paper, titled ‘The fibrin plate method for estimating fibrinolytic activity,’ presented a more standardized and refined version of this technique, enhancing its reproducibility and accuracy. They meticulously adjusted variables such as fibrinogen concentration and incubation conditions, thereby optimising the assay for broader applications across various fibrinolytic enzymes. Figure 1. Fibrin Plate Method for Fibrinolytic Activity Assessment. Illustration of the fibrin plate method as introduced by Permin in 1947 and standardised by Astrup & Müllertz in 1952. This technique involves the degradation of a fibrin clot by plasmin, where the extent of fibrinolysis is indicated by a clear zone, whose radius is proportional to the enzymatic activity present. Concurrently, we see the development of ‘Immuno-electrophoretic-analysis’ where components of serum are electrophoretically resolved in an agar gel matrix and are then exposed to specific antibodies allowing the detection of serum components (Grabar & Williams, 1953). Further refinement of this method, particularly through J. J. Scheidegger's slide gel electrophoresis in 1955, catalysed the foundational concepts for modern zymography. This method involved the electrophoretic separation of serum components followed by the diffusion of specific antibodies against these antigens, leading to the formation of distinct ‘precipitate arcs’ within the gel. This technique not only enabled the localisation of antigens within a gel matrix but also allowed for the determination of their electrophoretic mobility. Figure 2. Immuno-electrophoretic-analysis, an Early Zymography Technique Utilising Slide Gel Electrophoresis. A schematic representation of slide gel electrophoresis for analysing plasmin, as first described by Grabar & Williams and later Scheidegger. This method integrates the principles of fibrin plate methodology with electrophoretic separation, paving the way for modern zymography techniques. Heimburger and Schwick’s seminal paper titled ‘Die Fibrinagar-Elektrophorese’ from 1962 on electrophoretically analysing plasmin, plasminogen, their activators and inhibitors, is the first instance of resemblance to modern day zymography. The authors allude to inspiration from Grabar & Williams (1953) and Astrup & Müllertz (1952). In their approach, fibrin is incorporated into a slide gel used for the electrophoretic separation of plasma. This method ingeniously allowed the observation of clear zones of lysis post-incubation at specific locations corresponding to the presence of enzymes such as trypsin, chymotrypsin, and plasmin, identified by distance of migration, marking a pivotal advancement in detecting and analysing proteases and their inhibitors in human plasma. In addressing technical challenges inherent to zymography, particularly in the context of substrate immobilisation within electrophoretic gel matrices, two principal questions arise. Firstly, the potential migration of a charged substrate under electrophoretic conditions could be a cause of concern. This issue is mitigated by the intrinsic properties of fibrin, which, when copolymerized with acrylamide and bisacrylamide, remains stationary even under an electric field. The resultant fibrin-acrylamide matrix, characterized by its opacity and insolubility, precludes ionic migration. Secondly, the challenge of preventing premature enzymatic activity during electrophoresis is addressed by conducting the process at a suboptimal temperature, specifically 4°C. This strategic temperature regulation effectively inhibits enzymatic action until the desired post-separation incubation at pH 8.0, a condition under which protease activity is reported to be optimal. The term ‘zymogram’, and by extension zymography, however was coined in 1957 by Robertson and Wexler. Their method involved separation of a protein mixture in a starch gel and visualising enzymes through histochemical methods, successfully demonstrating enzyme activity retention within a hydrogel matrix, a concept foundational to zymography. (Hunter & Markert, 1957). The authors advocated it as a general method to study the enzymatic composition of tissues. Later this method was advocated as a method to discover new enzymes (Maravolo et al., 1967). Consequent to the development of PAGE as a method of resolving proteins, Hochstraßer & Schorn (1974) incorporated gelatin and azocaesin, which would function as the protease substrate into a Native PAG. This approach, which employs negative protein staining post-electrophoresis, has significantly influenced contemporary zymographic techniques, closely resembling current practices in terms of substrate incorporation and staining methodology. Interestingly, the foundational work of Heimburger and Schwick in 1962, which demonstrated a similar principle in the analysis of plasmin, plasminogen, and their activators and inhibitors, was not cited in Hochstraßer and Schorn's paper. This omission is intriguing, given the similarity in the underlying principles of zymography between the two studies, suggesting a potential underappreciation of Heimburger and Schwick's contributions at the time. Granelli-Piperno and Reich's 1978 exploration of extracellular proteolytic reactions using SDS-PAGE, later described as ‘overlay zymography,’ represents a significant leap in addressing the technical challenges of zymography. Their innovation was grounded in the observation that some serine proteases could be reversibly inhibited by SDS, effectively addressing the necessity of running electrophoresis at low temperatures or using pH levels that deactivate enzymes. This approach allowed for the removal of SDS from the PAGE gel through anionic detergent washing, as described by Converse and Papermaster in 1975. The advancements by Heussen and Dowdle in 1980 further solidified the role of SDS-PAGE in zymography. Their technique for detecting plasminogen-dependent and independent proteases in SDS-PAG, copolymerized with gelatin as a substrate, provided a refined approach to enzyme analysis. Notably, they regarded Reich's overlay method as complementary rather than substitutive, due to its limitations in real-time kinetic analysis of enzymatic activity. The rapid and widespread acceptance of SDS-based transient inactivation of enzymes, combined with linearization and negative charge distribution correlating mobility with molecular weight, as detailed by Shapiro et al. in 1967, is unsurprising. Further developments in zymography have been on the basis of this study, as described in the beginning of this section, which as of writing this review has 2455 citations well distributed over the years, testifying to its enduring impact and relevance in the field of biochemical research.
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