Quantifying the Modulation of Elastase Enzyme Activity Through Colorimetric Analysis

Summary

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

Abstract

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.

Introduction

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. 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 activity^{7,10,11,12,13}. This assay relies on the enzyme'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.

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)

1. Weigh the corresponding Tris base using an analytical balance.
2. Transfer the Tris base to a beaker and add deionized water using a graduated cylinder.
3. Stir the solution with a magnetic stirrer until the Tris base is completely dissolved.
4. Adjust the pH to 8.0 by adding 4 N HCl dropwise. Use a pH meter to monitor the pH.
5. Once the desired pH is reached, transfer the solution to a volumetric flask and fill to the desired volume mark with deionized water.
6. Transfer the buffer to a labeled storage bottle and store it at room temperature until use.

2. Sample preparation

1. Accurately weigh the required amount of the samples using an analytical balance. For this study, 1 mg of each sample is sufficient.
2. Dissolve the samples in RB to the desired concentration. Different sample concentrations can be tested, with 1 mg/mL being a good reference.
3. Use a vortex mixer to ensure the samples are fully dissolved.
4. 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.

3. Preparation of elastase enzyme

1. Prepare a working solution of elastase in RB at a final concentration of 10 µg/mL. Use the following equation to calculate the required volume of stock solution:
   Volume of stock (µL) = Desired final volume (µL) x Desired final concentration (µg/mL)/Stock concentration (µg/mL)
2. 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.

4. Preparation of the SANA substrate

1. 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 (µL) × Desired final concentration (mM) × Molecular weight (mg/mmol)/1000
2. Protect the solution from light and store it at 4 °C until use.

5. Preparation of the phenylmethylsulfonyl fluoride (PMSF) stock solution

1. Prepare a 100 mM of PMSF stock solution in isopropanol.
2. Store the prepared PMSF solution on ice until ready for use.
   NOTE: Aliquot the PMSF solution into smaller volumes (e.g., 100 µL) and store at -20 °C to avoid freeze-thaw cycles.

6. Setting up the assay

1. Prepare the following solutions in triplicate in microcentrifuge tubes:
   1. Negative control: Pipette 800 µL of RB.
   2. Vehicle control: Add 600 µL of RB and 200 µL of the solvent used to dissolve the sample.
   3. Positive inhibition control: Add 24 µL of PMSF stock solution and 776 µL of RB.
   4. Isopropanol control: Add 24 µL of isopropanol and 776 µL of RB.
   5. Samples: Pipette 200 µL of the prepared sample solution and add 600 µL of RB.
   6. Color controls (if needed): Pipette 200 µL of the sample solution and add 800 µL of RB.
2. Add 100 µL of the elastase solution (10 µg/mL) to each tube, except for the color controls.
3. Incubate all tubes at room temperature for 20 min.
4. Add 100 µL of SANA substrate solution (0.8 mM) to each tube, except for the color controls.
5. Mix the solutions thoroughly by gently inverting the tubes several times. Transfer 300 µL from each tube to a 96-well plate, ensuring triplicate measurements for each sample.
6. 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.

7. Data analysis

1. Normalize the results by setting the positive inhibition control (PMSF) as 100% inhibition and the negative control (RB only) as 0% inhibition.
2. Calculate the percentage of inhibition for each sample using the following formula:
   % Inhibition = 100 - [(Sample absorbance - Vehicle control absorbance - Color control absorbance)/(Negative control absorbance - Positive control absorbance - Isopropanol control absorbance)] × 100
3. Use graphing software to plot the inhibition percentages for each sample concentration against the sample concentration to visualize the inhibitory effect.

Results

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...

Discussion

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...

Materials

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