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.

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

652 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.

Video: Quantifying the Modulation of Elastase Enzyme Activity Through Colorimetric Analysis

Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical principle, with fiber-optic probes acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation.
Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments&#39; ability to provide quality kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular interaction model.

We describe here protocols for the measurement of antibody-antigen binding affinity and kinetics using four commonly used biosensor platforms.

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

We describe a modified small-scale extraction and colorimetric assays of lactate and pyruvate in the nematode C. elegans. When utilizing commercial assay kits, the technical development of their sensitivity and accuracy is important. Protein precipitation in extraction is the most critical step for the quantitative determination of intracellular metabolites.

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom body (MB) is a higher brain center proposed to be the neural substrate of complex honeybee behaviors. Although previous studies identified genes and proteins that are differentially expressed in the MBs and other brain regions, the activities of the proteins in each region are not yet fully understood. To reveal the functions of these proteins in the brain, pharmacologic analysis is a feasible approach, but it is first necessary to confirm that pharmacologic manipulations indeed alter the protein activity in these brain regions.
We previously identified a higher expression of genes encoding phospholipase C (PLC) in the MBs than in other brain regions, and pharmacologically assessed the involvement of PLC in honeybee behavior. In that study, we biochemically tested two pharmacologic agents and confirmed that they decreased PLC activity in the MBs and other brain regions. Here, we present a detailed description of how to detect PLC activity in honeybee brain homogenate. In this assay system, homogenates derived from different brain regions are reacted with a synthetic fluorogenic substrate, and fluorescence resulting from PLC activity is quantified and compared between brain regions. We also describe our evaluation of the inhibitory effects of certain drugs on PLC activity using the same system. Although this system is likely affected by other endogenous fluorescence compounds and/or the absorbance of the assay components and tissues, the measurement of PLC activity using this system is safer and easier than that using the traditional assay, which requires radiolabeled substrates. The simple procedure and manipulations allow us to examine PLC activity in the brains and other tissues of honeybees involved in different social tasks.

To test the inhibitory effects of pharmacologic agents on phospholipase C (PLC) in different regions of the honeybee brain, we present a biochemical assay to measure PLC activity in those regions. This assay could be useful for comparing PLC activity among tissues, as well as among bees exhibiting different behaviors.

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom ...

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of physiological processes. Mitochondrial dysfunction is a primary cause of a number of metabolic and neurodegenerative diseases. Intact mitochondria are critical for their proper functioning. The enzyme citrate synthase is localized in the mitochondrial matrix and thus can be used as a quantitative enzyme marker of intact mitochondrial mass. Given that many molecules and pathways that have important functions in mitochondria are highly conserved between humans and Drosophila, and that an array of powerful genetic tools are available in Drosophila, Drosophila serves as a good model system for studying mitochondrial function. Here, we present a protocol for fast and simple measurement of citrate synthase activity in tissue homogenate from adult flies without isolating mitochondria. This protocol is also suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues.

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

We present a protocol for a colorimetric assay of citrate synthase activity for quantification of intact mitochondrial mass in Drosophila tissue homogenates.

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

Proteomic Profile of EPS-Urine through FASP Digestion and Data-Independent Analysis

Filter-aided sample protocol (FASP) is widely used for proteomics sample preparation because it allows to concentrate diluted samples and it is compatible with a wide variety of detergents. Bottom-up proteomics workflows like FASP increasingly rely on LC-MS/MS methods performed in data-independent analysis (DIA) mode, a scanning method that allows deep proteome coverage and low incidence of missing values. 
In this report, we will provide the details of a workflow that combines a FASP protocol, a double StageTip purification step and LC-MS/MS in DIA mode for urinary proteome mapping. As a model sample, we analyzed expressed prostatic secretions (EPS)-urine, a sample collected after a digital rectal exam (DRE), which is of interest in prostate cancer biomarker discovery studies.

Here, we present an optimized on-filter digestion protocol with detailed information about the following: protein digestion, peptide purification and data independent acquisition analysis. This strategy is applied to the analysis of expressed prostatic secretions-urine samples and allows high proteome coverage and low missing value label-free profiling of the urinary proteome.

Filter-aided sample protocol (FASP) is widely used for proteomics sample preparation because it allows to concentrate diluted samples and it is compatible ...

Modeling an Enzyme Active Site using Molecular Visualization Freeware

Biomolecular visualization skills are paramount to understanding key concepts in the biological sciences, such as structure-function relationships and molecular interactions. Various programs allow a learner to manipulate 3D structures, and biomolecular modeling promotes active learning, builds computational skills, and bridges the gap between two dimensional textbook images and the three dimensions of life. A critical skill in this area is to model a protein active site, displaying parts of the macromolecule that can interact with a small molecule, or ligand, in a way that shows binding interactions. In this protocol, we describe this process using four freely available macromolecular modeling programs: iCn3D, Jmol/JSmol, PyMOL, and UCSF ChimeraX. This guide is intended for students seeking to learn the basics of a specific program, as well as instructors incorporating biomolecular modeling into their curriculum. The protocol enables the user to model an active site using a specific visualization program, or to sample several of the free programs available. The model chosen for this protocol is human glucokinase, an isoform of the enzyme hexokinase, which catalyzes the first step of glycolysis. The enzyme is bound to one of its substrates, as well as a non-reactive substrate analog, which allows the user to analyze interactions in the catalytic complex.

A key skill in biomolecular modeling is displaying and annotating active sites in proteins. This technique is demonstrated using four popular free programs for macromolecular visualization: iCn3D, Jmol, PyMOL, and UCSF ChimeraX.

Biomolecular visualization skills are paramount to understanding key concepts in the biological sciences, such as structure-function relationships and ...

Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ever-increasing share of published structural data. Recent advances in TEM technology and automation have boosted both the speed of data collection and quality of acquired images while simultaneously decreasing the required level of expertise for obtaining cryo-EM maps at sub-3 &#197; resolutions. While most of such high-resolution structures have been obtained using state-of-the-art 300 kV cryo-TEM systems, high-resolution structures can be also obtained with 200 kV cryo-TEM systems, especially when equipped with an energy filter. Additionally, automation of microscope alignments and data collection with real-time image quality assessment reduces system complexity and assures optimal microscope settings, resulting in increased yield of high-quality images and overall throughput of data collection. This protocol demonstrates the implementation of recent technological advances and automation features on a 200 kV cryo-transmission electron microscope and shows how to collect data for the reconstruction of 3D maps that are sufficient for de novo atomic model building. We focus on best practices, critical variables, and common issues that must be considered to enable the routine collection of such high-resolution cryo-EM datasets. Particularly the following essential topics are reviewed in detail: i) automation of microscope alignments, ii) selection of suitable areas for data acquisition, iii) optimal optical parameters for high-quality, high-throughput data collection, iv) energy filter tuning for zero-loss imaging, and v) data management and quality assessment. Application of the best practices and improvement of achievable resolution using an energy filter will be demonstrated on the example of apo-ferritin that was reconstructed to 1.6 &#197;, and Thermoplasma acidophilum 20S proteasome reconstructed to 2.1-&#197; resolution using a 200 kV TEM equipped with an energy filter and a direct electron detector.

High-resolution cryo-EM maps of macromolecules can be also achieved by using 200 kV TEM microscopes. This protocol shows the best practices for setting accurate optics alignments, data acquisition schemes, and selection of imaging areas that are all essential for the successful collection of high-resolution datasets using a 200 kV TEM.

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ...

Purifying a Fibrinolytic Enzyme from Sipunculus nudus via Affinity Chromatography

Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus

The fibrinolytic enzyme from Sipunculus nudus (sFE) is a novel fibrinolytic agent that can both activate plasminogen into plasmin and degrade fibrin directly, showing great advantages over traditional thrombolytic agents. However, due to the lack of structural information, all the purification programs for sFE are based on multistep chromatography purifications, which are too complicated and costly. Here, an affinity purification protocol of sFE is developed for the first time based on a crystal structure of sFE; it includes preparation of the crude sample and the lysine/arginine-agarose matrix affinity chromatography column, affinity purification, and characterization of the purified sFE. Following this protocol, a batch of sFE can be purified within 1 day. Moreover, the purity and activity of the purified sFE increases to 92% and&#160;19,200 U/mL,&#160;respectively. Thus, this is a simple, inexpensive, and efficient approach for sFE purification. The development of this protocol is of great significance for the further utilization of sFE and other similar agents.

Author Spotlight: Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus  

Here, we present an affinity purification method of a fibrinolytic enzyme from Sipunculus nudus that is simple, inexpensive, and efficient.

The fibrinolytic enzyme from Sipunculus nudus (sFE) is a novel fibrinolytic agent that can both activate plasminogen into plasmin and degrade fibrin ...