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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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.

Quantifying the Modulation of Elastase Enzyme Activity Through Colorimetric Analysis

The scope of our research focuses on the development of natural reactive compounds. So using an in vitro method, we aim to evaluate the activity of elastase, an enzyme involved in the degradation of elastin and the loss of elasticity, in order to restore the homeostasis of the tissue. Quantifying natural plant extracts, the activity, presents several challenges, mainly related to their complex chemical composition and their often strong coloration that can interfere with common measurements.Developing reliable adaptive protocols is vital for advancing this research field. Our study offers several benefits to the fields, including simplicity, sensitivity, fast result of tension, and adaptability to different research needs. To begin, weigh the appropriate amount of Tris base using an analytical balance and transfer the weighed Tris base to a beaker.Using a graduated cylinder, add deionized water to the beaker. Stir the solution with a magnetic stirrer until the Tris base is completely dissolved. Then adjust the solution's pH to eight using four normal hydrochloric acid.Once the desired pH is reached, transfer the solution to a volumetric flask. Using deionized water, fill the flask to the marked volume. Transfer the prepared buffer to a labeled storage bottle and store it at room temperature.For sample preparation, accurately weigh the required amount of sample using an analytical balance. Dissolve the sample in the prepared reaction buffer to achieve the desired concentration. Use a vortex mixer to fully dissolve the sample.And store the prepared sample on ice. Next, prepare a working solution of elastase enzyme in reaction buffer to a final concentration of 10 micrograms per milliliter. Store the elastase solution on ice until ready for use.Now prepare a 0.8 millimolar SANA solution in reaction buffer. Protect the solution from light and store it at four degrees Celsius until use. For the phenylmethanesulfonyl fluoride, or PMSF stock, prepare a 100 millimolar PMSF solution in isopropanol.Store the prepared solution on ice. Prepare all the reactions in triplicate using microcentrifuge tubes. Incubate all the tubes at room temperature for 20 minutes.Then add 100 microliters of the SANA substrate solution to each tube, except for the color controls. Gently invert the tubes several times to mix the samples and transfer 300 microliters from each tube to a 96-well plate, ensuring triplicate measurements for each sample. Finally, place the 96-well plate in a microplate reader set to 410 nanometers, and measure the absorbance periodically, every minute for 20 minutes, or until the signal stabilizes at room temperature.Extract one showed weak but significant elastase inhibitory activity at concentrations of one, 0.75, and 0.5 milligrams per milliliter, with no significant effect at 0.25 milligrams per milliliter. Extract two demonstrated high elastase inhibitory activity at all tested concentrations, with inhibition levels similar to the positive control at one, 0.75, and 0.5 milligrams per milliliter.

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...

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 ...

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JoVE Journal

Biochemistry

453 Views.  Miguel Hernández University (UMH).  This protocol describes the development of a colorimetric assay method for determining the ability of compounds to inhibit or activate elastase activity.

Measuring Elastase Modulation: A Colorimetric Assay for Therapeutic Insights

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