Fluorescent protein gel assays help characterize snake venom toxins and evaluate potential therapeutics

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In recent work, Bittenbinder et al developed new assays to study the proteolytic activity of snake venom and evaluate potential inhibitors. The assays take advantage of fluorescently labeled ECM proteins to characterize the proteolytic profiles of different snake venoms, assess kinetics and inhibition of proteolysis, and identify the proteins responsible for proteolysis. Fluorescent proteins were separated on SDS-PAGE and detected using the Azure 400 Imaging System (AZI400-01).

New assay to characterize proteolytic activities in venom

The authors from the Naturalis Biodiversity Center developed an assay to characterize the proteolytic activities present in the venom of eight medically relevant snakes against six ECM substrates (gelatin, collagen, elastin, fibronectin, laminin, and hyaluronic acid). For this assay, dye-quenched (DQ) fluorescently labeled ECM substrates were incubated with each snake venom. The DQ substrates are highly labeled such that their fluorescence is quenched until they are digested; once the proteins are digested, the resulting peptides fluoresce brightly. These labeled substrates are often used to study proteolysis by following the increase of fluorescence in the reaction.

In the present work, the authors ran the digestion reaction products on a gel to separate the products and visually assess which snake venoms were able to digest each ECM component. The gels were imaged on the Azure 400 using 472 nm or 524 nm excitation, depending on the fluorescent label (Figure 1D).

Fluorescently labeled substrates using Azure 400
Panel from Figure 1D from Bittenbinder et al (2023). Monitoring snake venom-induced extracellular matrix degradation and identifying proteolytically active venom toxins using fluorescently labeled substrates. Licensed under CC BY 4.0. The figure shows the ability of eight snake venom samples to degrade fluo-hyaluronic acid. Activity across the different types of snake venoms ranges from very high (D. polylepis, DePo) to very low (N. naja, NaNa).

The assay provides a powerful way to scan multiple venoms for activity against multiple ECM targets. By stopping the reactions at various times, the authors were able to follow the kinetics of substrate degradation. In addition, the assay allowed them to study whether known protease inhibitors could block the degradation of ECM substrates, which could be applied to identifying or evaluating novel compounds for inhibitory activity.

A new fluo-zymography assay

A second type of assay, a new fluo-zymography assay (Figure 5), was developed to identify the proteins in the venom responsible for degrading each substrate. In conventional zymography assays, gelatin or collagen was included in SDS-PAGE gels. Venom proteins were run in the gels, then incubated in activity buffer overnight.

Figure 5 from Bittenbinder et al (2023). Monitoring snake venom-induced extracellular matrix degradation and identifying proteolytically active venom toxins using fluorescently labeled substrates. Licensed under CC BY 4.0. Figure 5 shows a fluo-zymography assay developed by the authors. Eight snake venom samples were separated on an SDS-PAGE gel containing DQ-gelatin (fluorescently labeled and quenched gelatin). After overnight incubation in activity buffer, fluorescent bands reveal the location of proteins that have proteolytic activity against gelatin. The authors cut out these bands and analyzed by LC-MS/MS to identify the toxin protein in each band.

Gels were stained with Coomassie Brilliant Blue. Proteins that degraded the gelatin or collagen appeared as clear bands in a dark background. The authors’ new fluo-zymography assay included fluorescently labeled (quenched) gelatin or collagen in the gel matrix. Bands representing proteins that degraded gelatin or collagen are brightly fluorescent (see second figure). Gels were again imaged using the Azure 400.

The fluo-zymography assay has an advantage over conventional zymography in requiring a lower substrate (gelatin or collagen) concentration, which could conserve substrate or make the assay possible when substrate amounts are limited.

What this research means for the future of potential therapeutics

The authors conclude that these new assays allow visualization and comparison of the proteolytic activity of snake venoms. The fluo-zymography assay offers a new, sensitive way, in combination with proteomics, to identify proteins in venom with proteolytic activity. The new assays could help identify and characterize both the toxins responsible for snakebite morbidity as well as new therapeutics to treat snake bite toxicity.

Background on the impact of snake bites

Snake bites kill as many as 137,880 people per year worldwide. About three times more people experience permanent disability from non-mortal bites1. Snake bite morbidity and mortality are attributable to the toxins in snake venom, most of which are proteins. These snake venom toxins cause paralysis, kidney failure, and fatal hemorrhage, as well as tissue damage, leading to permanent disability and amputation. In fact, three of the four major classes of snake venom, including snake venom serine proteases, cause tissue damage. Targets for toxin proteases include the proteins that make up the extracellular matrix (ECM). The ECM provides structural support to tissue and is important for cell viability; degrading the ECM leads to tissue damage and bleeding.

Used in this study: the Azure 400 Imager

Azure 400 Visible fluorescent imaging system
The Azure 400 is capable of three-channel visible fluorescence detection, which enables sensitive multiplex detection of Western blots, fluorescent biomolecules and Cy2/Cy3/Cy5 or similar fluorochromes. This fluorescent imager allows users to simultaneously image and quantify up to three different targets.

The Azure 400 is a fluorescent imager that provides high-resolution imaging is ideal for publication purposes, as well as higher pixel well cap for higher dynamic range.

It is a flexible fluorescent imager that enables three-color fluorescent detection for dyes in the visible range. With the Azure 400, you can simultaneously image and quantify up to three different targets. This fluorescent imager is capable of three-channel visible fluorescence detection, and enables sensitive multiplex detection of Western blots, fluorescent biomolecules and Cy2/Cy3/Cy5 or similar fluorochromes.

More research done with the Azure 400

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Two scientists looking at multiplex fluorescent Western blot on Azure 600 Western blot imager
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SOURCES

  1. Snakebite envenoming. The World Health Organization website. Published September 12, 2023. Accessed February 5, 2024. https://www.who.int/news-room/fact-sheets/detail/snakebite-envenoming.
  2. Bittenbinder MA, Bergkamp ND, Slagboom J, et al. Monitoring snake venom-induced extracellular matrix degradation and identifying proteolytically active venom toxins using fluorescently labeled substrates. Biology. 2023;12(6):765.

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