This research was funded by the Spanish Ministry of Science and Innovation (MCIN/AEI/10.13039/501100011033/FEDER, UE; projects: RTI2018-096724-B-C21, TED2021-129932B-C21, and PID2021-125188OB-C32) and the Generalitat Valenciana (PROMETEO/2021/059). This work was also supported by the Official Funding Agency for Biomedical Research of the Spanish Government, Institute of Health Carlos III (ISCIII) through CIBEROBN (CB12/03/30038), Agencia Valenciana de la Innovaci&#243;n: INNEST/2022/103; which is co-funded by the European Regional Development Fund. E.B.-C and M.H.-L. have been supported by the Requalification of the Spanish University System for 2021/2023 grant. F.J.&#193;.-M. has been supported by Margarita Salas Grants for the training of young doctors 2021/2023. We would like to extend our heartfelt gratitude to the administrative and technical support staff whose unwavering assistance was invaluable in the development of this protocol.

The details of the reagents and the equipment used for this study are listed in the Table of Materials.
1. Preparation of 0.2 M Tris base reaction buffer (RB)
<ol>
	<li>Weigh the corresponding Tris base using an analytical balance.</li>
	<li>Transfer the Tris base to a beaker and add deionized water using a graduated cylinder.</li>
	<li>Stir the solution with a magnetic stirrer until the Tris base is completely dissolved.</li>
	<li>Adjust the pH to 8.0 by adding 4 N HCl dropwise. Use a pH meter to monitor the pH.</li>
	<li>Once the desired pH is reached, transfer the solution to a volumetric flask and fill to the desired volume mark with deionized water.</li>
	<li>Transfer the buffer to a labeled storage bottle and store it at room temperature until use.</li>
</ol>
2. Sample preparation
<ol>
	<li>Accurately weigh the required amount of the samples using an analytical balance. For this study, 1 mg of each sample is sufficient.</li>
	<li>Dissolve the samples in RB to the desired concentration. Different sample concentrations can be tested, with 1 mg/mL being a good reference.</li>
	<li>Use a vortex mixer to ensure the samples are fully dissolved.</li>
	<li>Store the prepared samples on ice until ready for use. 
	NOTE: If the sample has a strong coloration that might interfere with absorbance readings, prepare color controls by following the same procedure but without adding elastase enzyme in later steps.</li>
</ol>
3. Preparation of elastase enzyme
<ol>
	<li>Prepare a working solution of elastase in RB at a final concentration of 10 &#181;g/mL. Use the following equation to calculate the required volume of stock solution: 
	Volume of stock (&#181;L) = Desired final volume (&#181;L) x Desired final concentration (&#181;g/mL)/Stock concentration (&#181;g/mL)</li>
	<li>Store the prepared elastase solution on ice until ready for use. 
	NOTE: Consider the specific activity of the enzyme as provided by the supplier, which may vary between batches. Also, enzyme activity may decrease after freezing; prepare fresh solutions when possible.</li>
</ol>
4. Preparation of the SANA substrate
<ol>
	<li>Prepare 0.8 mM of SANA solution by dissolving the appropriate amount of SANA powder in RB. Use the following equation: 
	Mass of SANA (mg) = Desired final volume (&#181;L) &#215; Desired final concentration (mM) &#215; Molecular weight (mg/mmol)/1000</li>
	<li>Protect the solution from light and store it at 4 &#176;C until use.</li>
</ol>
5. Preparation of the phenylmethylsulfonyl fluoride (PMSF) stock solution
<ol>
	<li>Prepare a 100 mM of PMSF stock solution in isopropanol.</li>
	<li>Store the prepared PMSF solution on ice until ready for use. 
	NOTE: Aliquot the PMSF solution into smaller volumes (e.g., 100 &#181;L) and store at -20 &#176;C to avoid freeze-thaw cycles.</li>
</ol>
6. Setting up the assay
<ol>
	<li>Prepare the following solutions in triplicate in microcentrifuge tubes:
	<ol>
		<li>Negative control: Pipette 800 &#181;L of RB.</li>
		<li>Vehicle control: Add 600 &#181;L of RB and 200 &#181;L of the solvent used to dissolve the sample.</li>
		<li>Positive inhibition control: Add 24 &#181;L of PMSF stock solution and 776 &#181;L of RB.</li>
		<li>Isopropanol control: Add 24 &#181;L of isopropanol and 776 &#181;L of RB.</li>
		<li>Samples: Pipette 200 &#181;L of the prepared sample solution and add 600 &#181;L of RB.</li>
		<li>Color controls (if needed): Pipette 200 &#181;L of the sample solution and add 800 &#181;L of RB.</li>
	</ol>
	</li>
	<li>Add 100 &#181;L of the elastase solution (10 &#181;g/mL) to each tube, except for the color controls.</li>
	<li>Incubate all tubes at room temperature for 20 min.</li>
	<li>Add 100 &#181;L of SANA substrate solution (0.8 mM) to each tube, except for the color controls.</li>
	<li>Mix the solutions thoroughly by gently inverting the tubes several times. Transfer 300 &#181;L from each tube to a 96-well plate, ensuring triplicate measurements for each sample.</li>
	<li>Place the 96-well plate into a microplate reader set to 410 nm. Measure the absorbance periodically, every minute, for 20 min or until the signal stabilizes. Set the reader to measure at room temperature. 
	NOTE: If the plate reader supports kinetic measurements, program it to automatically take readings at set intervals. Otherwise, manually record absorbance at the desired time points.</li>
</ol>
7. Data analysis
<ol>
	<li>Normalize the results by setting the positive inhibition control (PMSF) as 100% inhibition and the negative control (RB only) as 0% inhibition.</li>
	<li>Calculate the percentage of inhibition for each sample using the following formula: 
	%&#160;Inhibition = 100 - [(Sample absorbance - Vehicle control absorbance - Color control absorbance)/(Negative control absorbance - Positive control absorbance - Isopropanol control absorbance)] &#215; 100</li>
	<li>Use graphing software to plot the inhibition percentages for each sample concentration against the sample concentration to visualize the inhibitory effect.</li>
</ol>

Measuring Elastase Modulation: A Colorimetric Assay for Therapeutic Insights

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The authors declare no conflicts of interest.

In the present method, the modulatory effects of phytochemicals on elastase enzymes are examined using a colorimetric assay. Elastase, a serine protease crucial for elastin degradation, plays a significant role in maintaining tissue elasticity in various organs. The colorimetric elastase assay described in this work offers a simple, sensitive, and rapid method for measuring elastase activity.
In this context, researchers have focused on modulators, such as plant extracts and pure phytochemical...

Elastase is a serine protease enzyme that plays a crucial role in breaking down elastin, a protein that provides elasticity to various tissues in the body, including the lungs, skin, and blood vessels. Elastase activity is tightly regulated to maintain tissue homeostasis, and dysregulation can lead to pathological and dermal conditions such as emphysema, atherosclerosis, and skin wrinkles1.
There are several types of elastases, each with specific characteristics and functions. Neutrophil elastases, produced by neutrophils, are important in the immune response and inflammation. These enzymes can degrade a wide variety of extracellular proteins and are involved in chronic inflammatory diseases2. Pancreatic elastases, on the other hand, play a role in protein digestion in the small intestine3. Distinguishing between these elastases is crucial for developing specific therapies for different diseases.
Healthy levels of elastin and pathways that regulate elastase activity help preserve the elasticity of the skin and prevent premature aging. Factors such as aging, UV radiation, inflammation, genetic predisposition, environmental pollutants, and nutrition significantly influence the activity and degradation of elastase4. An emerging area of interest is the study of elastokines, bioactive fragments generated by the degradation of elastin by elastase. These molecules can induce significant biological effects, including increased inflammation, elastic fiber calcification, and lipid deposition, among others5. Elastokines may influence the progression of diseases associated with elastin degradation and offer a potential target for new therapeutic interventions (Figure 1).
Some compounds, such as naturally occurring phytochemicals, have gained significant attention for their potential therapeutic and cosmetic effects, including their ability to modulate elastase activity6. For instance, quercetin, a flavonoid found in apples and onions, has been shown to effectively inhibit elastase activity, which contributes to its anti-inflammatory and anti-aging effects7. Curcumin, the bioactive compound in turmeric, is another well-studied phytochemical that exhibits elastase inhibition, offering protective effects against skin aging and inflammation8. Additionally, epigallocatechin gallate (EGCG), the primary catechin in green tea, has demonstrated potent elastase inhibitory activity, making it a valuable compound for skin care formulations aimed at preserving skin elasticity9.&#160;These examples underscore the potential of phytochemicals as natural elastase inhibitors, providing a foundation for the development of new therapeutic and cosmetic products.
Currently, the colorimetric elastase assay is a widely used method for measuring elastase activity7,10,11,12,13. This assay relies on the enzyme&#39;s ability to hydrolyze a specific substrate, N-succinyl-(Ala)3-nitroanilide (SANA), into succinylamino acids and p-nitroaniline (pNA). pNA is a yellow-colored chromophore that can be easily detected at 410 nm using a spectrophotometer (Figure 2). The rate of pNA production is directly proportional to the elastase activity in sample14.
This method has a wide range of applications in various research fields. Through this method, researchers can rapidly identify the ability of compounds to modulate elastase activity, investigate the mechanisms of action of elastase inhibitors, and evaluate the efficacy of these inhibitors in cellular and animal models of elastase-related diseases. Additionally, the assay can be used to study different mechanisms of inhibition, such as competitive or non-competitive inhibition, providing valuable information on how natural or synthetic compounds modulate elastase activity.
The elastase activity modulation assay offers several advantages over other methods for measuring elastase activity. It is simple and easy to perform, requires minimal technical expertise, and can be conducted in a standard laboratory setting15. In addition, the assay is highly sensitive and can detect small changes in elastase activity. The assay provides rapid and quantitative results, allowing for efficient data analysis. Moreover, it can be adapted to various formats, including high-throughput screening and kinetic studies16.
However, the assay also has several limitations, such as low substrate specificity (as it is specific for elastase), and susceptibility to interference from other components in the sample, such as colored compounds or inhibitors of pNA hydrolysis. Therefore, researchers must consider these limitations and use complementary methods to comprehensively investigate the mechanisms underlying the action of elastase inhibitors17.
There are elastase activity monitoring alternative methods, such as zymography, which offers an excellent tool for identifying and differentiating various elastase isoforms, which is crucial when studying the specific contributions of different elastase subtypes to a particular disease process. However, zymography is a semi-quantitative technique and requires additional steps for visualization; thus, compared to the spectrophotometric method, zymography is less efficient for high-throughput analysis18. Fluorometric assays offer increased sensitivity to the spectrophotometric method, providing lower detection limits. This allows for a more sensitive analysis of elastase activity and modulator interactions, providing a more complete picture of enzymatic processes19. However, fluorometric assays require specialized instrumentation and can be susceptible to interference from certain compounds in biological samples. Radiometric assays achieve exceptional sensitivity, making them ideal for the detection of very low levels of elastase activity. However, the use of radioactive materials necessitates specialized equipment, stringent safety protocols, and proper waste disposal procedures, which pose logistical challenges and safety concerns20. Immunoassays stand out for their versatility and can be used to measure elastase activity directly or quantify elastase-inhibitor complexes, providing insights into inhibitor efficacy. Additionally, immunoassays can be adapted to work with complex biological samples, such as tissue homogenates, unlike the SANA method. However, developing and validating immunoassays can be time-consuming and require specific antibodies, potentially leading to higher costs than those of simpler spectrophotometric approaches21.
The colorimetric elastase activity modulation assay is a valuable tool for investigating the ability of any compound to modify elastase activity. Due to its simplicity, sensitivity, and adaptability, the method is widely used in various research settings. However, researchers must consider the limitations of the assay and use complementary methods to comprehensively determine the mechanisms underlying the activity of elastase inhibitors.

<table><tbody><tr><td>96 Well Cell Culture Plate</td><td>Corning Incorporated</td><td>3599</td><td>Flat bottom with lid, polystyrene</td></tr><tr><td>Cell Imaging Multimode Reader</td><td>Agilent</td><td>BioTek Cytation 1</td><td>Used with Gen5 software</td></tr><tr><td>Elastase From Porcine Pancreas</td><td>Sigma-Aldrich</td><td>E7885</td><td>CAS 39445-21-1; 25,9 kDa</td></tr><tr><td>Isopropanol 99.5%</td><td>Fisher Scientific</td><td>AC184130010</td><td>CAS 67-63-0; C3H8O; 60.10 g/mol</td></tr><tr><td>N-Succinil-(Ala)3-nitroanilide</td><td>Sigma-Aldrich</td><td>S4760</td><td>CAS 52299-14-6; C19H25N5O8 ; 451.43 g/mol</td></tr><tr><td>pH Meter</td><td>Hach Lange</td><td>sensION+ PH31</td><td>With magnetic stirrer and sensor holder</td></tr><tr><td>Phenylmethanesulfonyl Fluoride</td><td>Sigma-Aldrich</td><td>P7626</td><td>CAS 329-98-6; C7H7FO2S; 174,19 g/mol</td></tr><tr><td>Tris For Molecular Biology</td><td>PanReac AppliChem</td><td>A2264</td><td>CAS 77-86-1; C4H11NO3; 121,14 g/mol</td></tr></tbody></table>

quantifying the modulation of elastase enzyme activity through colorimetric analysis

Elastase, a serine protease, plays an essential role in elastin degradation. Elastin is an extracellular protein that helps maintain tissue elasticity in the lungs, skin, and blood vessels. Tight regulation of elastase activity is crucial for tissue homeostasis, as dysregulation can contribute to pathologies such as emphysema, wrinkles, and atherosclerosis. Some compounds, such as naturally occurring phytochemicals, have shown potential for therapeutic intervention and have attracted significant interest. Elucidating the modulatory effects of different compounds on elastase, whether inhibitory or stimulatory, is crucial for developing novel therapeutic and cosmetic strategies targeting elastase-associated disorders. A widely accepted method for measuring elastase activity is the colorimetric elastase assay. In this assay, a specific substrate is used to break down elastase, releasing a detectable yellow compound, p-nitroaniline (pNA). The amount of pNA produced reflects elastase activity in the sample and can be measured by colorimetry. This assay offers several benefits, including simplicity, high sensitivity, rapid results, and adaptability to various research needs. The colorimetric elastase assay remains a valuable tool for studying how compounds impact elastase activity. Due to its ease of use and effectiveness, this assay is a cornerstone of research in this field.

Once the protocol is completed, the absorbance data necessary to perform the pertinent calculations and quantify the capacity of samples to modulate elastase activity can be obtained. Figure 3 highlights the location of the wells with the different controls and samples. In the case of colored samples, such as the one used in this example, it is necessary to add color controls to minimize spectrophotometric interference, as color can interfere with the measurement of the yellow color of pNA a...

Quantifying the Modulation of Elastase Enzyme Activity Through Colorimetric Analysis

This protocol describes the development of a colorimetric assay method for determining the ability of compounds to inhibit or activate elastase activity.

Elastase, a serine protease, plays an essential role in elastin degradation. Elastin is an extracellular protein that helps maintain tissue elasticity in ...

quantifying-modulation-elastase-enzyme-activity-through-colorimetric

Research

JoVE Journal

Biochemistry

907 Views.  Miguel Hernández University (UMH). De reikwijdte van ons onderzoek richt zich op de ontwikkeling van natuurlijke reactieve verbindingen. Met behulp van een in vitro-methode willen we de activiteit van elastase evalueren, een enzym dat betrokken is bij de afbraak van elastine en het verlies van elasticiteit, om de homeostase van het weefsel te herstellen. Het kwantificeren van natuurlijke plantenextracten, de activiteit, brengt verschillende uitdagingen met zich mee, voornamelijk in verband met...

Video: Kwantificering van de modulatie van de activiteit van het elastase-enzym door middel van colorimetrische analyse

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Microplate based procedures are described for the colorimetric or fluorometric analysis of extracellular enzyme activity. These procedures allow for the rapid assay of such activity in large numbers of environmental samples within a manageable time frame.

Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

Environment

Detection of Protease Activity by Fluorescent Peptide Zymography

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using electrophoresis through a resolving gel embedded with a degradable substrate. This method differs from traditional gel zymography in that a quenched fluorogenic peptide is covalently incorporated into the resolving gel instead of full length proteins, such as gelatin or casein. Use of the fluorogenic peptides enables direct detection of proteolytic activity without additional staining steps. Enzymes within the biological samples cleave the quenched fluorogenic peptide, resulting in an increase in fluorescence. The fluorescent signal in the gels is then imaged with a standard fluorescent gel scanner and quantified using densitometry. The use of peptides as the degradable substrate greatly expands the possible proteases detectable with zymographic techniques.

Here, we present a detailed protocol for a modified zymographic technique in which fluorescent peptides are used as the degradable substrate in place of native proteins. Electrophoresis of biological samples in fluorescent peptide zymograms enables detection of a wider range of proteases than previous zymographic techniques.

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using ...

Cancer Research

Ecotoxicological Methodologies to Evaluate Biomarkers at Different Scales in Neotropical Anurans

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that improve not only the interpretation of the effects of environmental stressors on organisms but also the determination of their possible impact. It is well known that the use of ecotoxicological biomarkers at different levels of organization allows for the prediction of the biological responses of organisms to environmental stressors, which is useful in environmental risk assessment. 
Nevertheless, it is necessary to consider the optimization of basic procedures, to generate historical data in control groups, and to employ specific bioassays to evaluate responses in organs and tissues in order to elucidate the nature and variation of the effects observed. Therefore, the present work aims to describe several ecotoxicological methodologies employed in all stages of neotropical anurans at different ecological levels and to validate them as useful biomarkers to be used both in wildlife and in laboratory conditions. In this work, these biomarkers were applied at the individual/organismic level (body condition index), histological/physiological level (histopathology, histometric, and pigmentary analyses), biochemical level (oxidative stress enzymes), and genetic level (direct and oxidative damage in DNA by comet assay). 
Although these methodologies have small variations or modifications depending on the species, these techniques provide effective biomarkers for evaluating the effect of xenobiotics on anurans, which possess certain characteristics that make them useful indicator species of aquatic and terrestrial ecosystems. In conclusion, the battery of biomarkers employed in the present study has proven to be adequate for estimating toxic responses in Neotropical anurans and can be further recommended as bioindicators for identifying the impact of pollutants on the aquatic ecosystems of the region. Finally, it is recommended to achieve the standardization of these important biomarkers for anurans in specific regions as well as to possibly include them in risk assessments and decision-making.

In this paper, standardized ecotoxicological methods for the evaluation of biomarkers in neotropical anuran species are presented. Specifically, this paper details several methodologies at different scales of ecotoxicological evaluation, such as the genetic, cellular-histological, biochemical, morphological, and individual levels.

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that ...

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

Biology

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Immunology and Infection

Colorimetric Analysis of Alkaline Phosphatase Activity in S. aureus Biofilm

Alkaline phosphatase (ALP) is a common enzyme expressed in both prokaryotic and eukaryotic cells. It catalyzes the hydrolysis of phosphate monoesters from many molecules at basic pH and plays an indispensable role in phosphate metabolism. In humans, eukaryotic ALP is one of the most frequently used enzymatic signals in diagnosing various diseases, such as cholestasis and rickets. In S. aureus, ALP is detected exclusively on the cell membrane; it is also expressed as a secretory form as well. Yet, little is known about its function in biofilm formation.
The purpose of this manuscript is to develop a quick and reliable assay to measure ALP activity in S. aureus biofilm that does not require protein isolation. Using p-nitrophenyl phosphate (pNPP) as a substrate, we measured ALP activity in S. aureus biofilm formed in 96-well tissue culture plates. Activity was based on the formation of the soluble reaction product measured by 405 nm absorbance. The high throughput nature of the 96 well tissue culture plate method provides a sensitive and reproducible method for ALP activity assays. The same experimental set up can also be extended to measure other extracellular molecular markers related to biofilm formation.

Colorimetric Analysis of Alkaline Phosphatase Activity in S. aureus Biofilm

In this manuscript, we established a high throughput method to quantify alkaline phosphatase activity in S. aureus biofilm culture from 96-well tissue culture plate

Alkaline phosphatase (ALP) is a common enzyme expressed in both prokaryotic and eukaryotic cells. It catalyzes the hydrolysis of phosphate monoesters from ...

Bioengineering

Monitoring Neutrophil Elastase and Cathepsin G Activity in Human Sputum Samples

Proteases are regulators of countless physiological processes and the precise investigation of their activities remains an intriguing biomedical challenge. Among the ~600 proteases encoded by the human genome, neutrophil serine proteases (NSPs) are thoroughly investigated for their involvement in the onset and progression of inflammatory conditions including respiratory diseases. Uniquely, secreted NSPs not only diffuse within extracellular fluids but also localize to plasma membranes. During neutrophil extracellular trap (NETs) formation, NSPs become an integral part of the secreted chromatin. Such complex behavior renders the understanding of NSPs pathophysiology a challenging task. Here, detailed protocols are shown to visualize, quantify and discriminate free and membrane-bound neutrophil elastase (NE) and cathepsin G (CG) activities in sputum samples. NE and CG are NSPs whose activities have pleiotropic roles in the pathogenesis of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD): they promote tissue remodeling, regulate downstream immune responses and correlate with lung disease severity. The protocols show how to separate fluid and cellular fraction, as well as the isolation of neutrophils from human sputum for enzymatic activity quantification via small-molecule F&#246;rster resonance energy transfer-based (FRET) reporters. To gather specific insights into the relative role of NE and CG activities, a FRET readout can be measured by different technologies: i) in vitro plate reader measurements allow for high-throughput and bulk detection of protease activity; ii) confocal microscopy spatiotemporally resolves membrane-bound activity at the cell surface; iii) small-molecule FRET flow cytometry enables for the rapid evaluation of anti-inflammatory treatments via single-cell protease activity quantification and phenotyping. The implementation of such methods opens the doors to explore NSPs pathobiology and their potential as biomarkers of disease severity for CF and COPD. Given their standardization potential, their robust readout and simplicity of transfer, the described techniques are immediately shareable for implementation across research and diagnostic laboratories.

The protocols herein described provide a guide to visualize and quantify the activity of neutrophil proteases in human sputum. The applications of such analysis span from the evaluation of anti-inflammatory treatments, to biomarker validation, drug screening and large cohort clinical studies.

Proteases are regulators of countless physiological processes and the precise investigation of their activities remains an intriguing biomedical ...

Zymography Gel Electrophoresis: An Electrophoretic Technique to Detect Matrix Metalloproteinases by Assessing the Enzymatic Activity

Source: Hu, X. et al., <a href="https://www.jove.com/v/2445">Detection of Functional Matrix Metalloproteinases by Zymography</a>. J. Vis. Exp. (2010)
This video demonstrates the zymography gel electrophoresis technique to assess the activity of matrix metalloproteinases (MMPs) present in cell culture supernatant. The active MMPs degrade the MMP-specific substrate embedded in a polyacrylamide gel, which appears as clear bands against a dark stained background following staining.

Source: Hu, X. et al., Detection of Functional Matrix Metalloproteinases by Zymography. J. Vis. Exp. (2010)
This video demonstrates the zymography gel ...

JoVE Encyclopedia of Experiments

Biological Techniques

Electrophoresis Techniques

An In Vitro Assay for Measuring Neutrophil Serine Protease Activity Using a Fluorescent Reporter

Source: Frey, D. L., et al. <a href="https://app.jove.com/v/62193">Monitoring Neutrophil Elastase and Cathepsin G Activity in Human Sputum Samples.</a> J. Vis. Exp. (2021).
This video demonstrates an in vitro assay to quantify neutrophil serine protease activity in sputum samples. The sample containing secreted protease is incubated with a fluorescent reporter bearing a recognition motif. Cleavage of the motif by the protease enables individual fluorescence emission by the donor and acceptor fluorophore of the reporter, indicating protease activity in the sample.

NOTE: The following protocols describe the sample preparation and the quantification of neutrophil serine proteases (NSPs) activity. The experimental ...

Immunology

Immunodiagnostics

Specialized Assays

Fluorescent Peptide Zymography: A Modified Technique to Detect Protease Activity Using Fluorogenic Substrates in Zymogram Gels

Source: Deshmukh, A. A. et al. <a href="https://www.jove.com/video/58938" target="_blank">Detection of Protease Activity by Fluorescent Peptide Zymography.</a> J. Vis. Exp. (2019)
In this video, we perform zymography, an electrophoretic technique, to analyze the proteolytic activity within biological samples.

Source: Deshmukh, A. A. et al. Detection of Protease Activity by Fluorescent Peptide Zymography. J. Vis. Exp. (2019)
In this video, we perform zymography, ...

Quantifying the Modulation of Elastase Enzyme Activity Through Colorimetric Analysis

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