PAL was funded by a Parkinson's UK Research Fellowship (grant F1002). This work was supported by a MRC New Investigator Research Grant (MR/L010933/1) and by MRC Programme grant MR/N026004/1 to PAL, and by BBSRC studentship BB/M017222/1 to JET. We thank the 2014-15 cohort of MPharm Part 1 students at the University of Reading for taking part in the first iteration of this practical, and subsequent cohorts for their feedback and input.

NOTE: The protocol below was developed to be held over the course of two hours (including completion of in-class worksheets) in a purpose-built teaching laboratory. The experimental steps were deliberately put together to exclude more detailed analysis of enzyme kinetics (carried out in the context of this course using model data); however &#8212; as noted in the discussion &#8212; the protocol provides a template for more advanced analyses depending upon time, facilities and student attainment level.
SAFETY: This practical should be carried out according to the rules of Good Chemical Laboratory Practice (GCLP) &#8212; personal protective equipment, including fastened laboratory coat, disposable gloves and safety glasses should be worn at all times in the laboratory.
1. Extracting the Enzyme
<ol>
	<li>Crush one lactase tablet, containing 200 mg of enzyme, to an even powder using a mortar and pestle.</li>
	<li>Place the resulting powder into a 15 mL tube labelled &#34;suspension&#34; and resuspend in 10 mL of 100 mM phosphate buffered saline (PBS).</li>
	<li>Vortex for 1 min to maximize enzyme extraction.</li>
	<li>Transfer 1 mL from the suspension into a 1.5 mL centrifuge tube and centrifuge for 1 min at 10,000 x g to sediment solid particles.</li>
	<li>Transfer 500 &#181;L of the supernatant to a clean 1.5 mL tube labelled &#34;Lactase Extract&#34;.</li>
</ol>
2. Observing the Colorimetric Reaction
<ol>
	<li>Place 390 &#181;L of 100 mM PBS into a 1.5 mL tube labelled &#34;Reaction A&#34;.</li>
	<li>Add 100 &#181;L of 5 mM ONPG solution and mix well by vortexing. 
	CAUTION: This practical uses ortho-Nitrophenol-beta-D-galactopyranoside (ONPG) as a substitute for lactose. Since ONPG is a phenolic compound, it should be handled with caution. In the event of skin contact with ONPG, the exposed skin should be washed immediately. Any spills should be wiped up immediately with paper towels to be disposed of into the appropriate waste stream.</li>
	<li>Add 10 &#181;L of extract into the reaction A tube and mix well by vortexing.</li>
	<li>Observe the reaction mixture for 5 minutes and note any colorimetric changes to the solution.</li>
</ol>
3. Measuring Enzyme Activity
<ol>
	<li>Set up two 1.5 mL tubes: one labelled &#34;Reaction B&#34;, one labelled &#34;Control&#34;.</li>
	<li>Add 390 &#181;L of 100 mM PBS to the reaction B tube, 400 &#181;L of 100 mM PBS to the control tube and then add 100 &#181;L of 5 mM ONPG to each tube.</li>
	<li>Mix the contents by vortexing.</li>
	<li>Add 10 &#181;L of lactase extract to the reaction B tube, mix by vortexing and allow the reaction to proceed for 1 min at room temperature.</li>
	<li>Once 1 min has elapsed, add 500 &#181;L of 1 M sodium carbonate to both tubes to inhibit the lactase enzyme by increasing the pH, thereby terminating the reaction.</li>
	<li>Transfer 500 &#181;L from each tube into clean spectrophotometer cuvettes and measure absorbance at 420 nm using the spectrophotometer.</li>
	<li>Note absorbance for reaction B and control samples, and then subtract the control value from the reaction value to derive the change in absorbance due to hydrolysis of ONPG in the presence of active enzyme.</li>
</ol>
4. Effect of Temperature on Enzyme Activity
<ol>
	<li>Set up three control tubes and three reaction tubes as described in section 3 and label these &#34;4 &#176;C&#34;, &#34;Room Temp&#34;, and &#34;37 &#176;C&#34;.</li>
	<li>Following addition of enzyme extract, incubate one tube on ice, one at room temperature, and one at 37 &#176;C (in a pre-heated water bath).</li>
	<li>Allow the reactions to proceed for 1 min and then terminate the reaction by adding 500 &#181;L of 1 M sodium carbonate to each tube.</li>
	<li>Measure the absorbance at 420 nm for each tube as described in section 3. Record the values, subtracting the control value from the reaction value in each case.</li>
</ol>

<ol><li>Berg, J. M., Tymoczko, J. L., Gatto, J. G., Stryer, L. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBerg%2C+JM+%22Biochemistry%22">Biochemistry</a>. , 8th, Palgrave Macmillan. (2015).</li>
<li>Jittam, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Red+seaweed+enzyme-catalyzed+bromination+of+bromophenol+red%3A+An+inquiry-based+kinetics+laboratory+experiment+for+undergraduates%5BTitle%5D)+AND+%22Biochemistry+and+Molecular+Biology+Education%22%5BJournal%5D)">Red seaweed enzyme-catalyzed bromination of bromophenol red: An inquiry-based kinetics laboratory experiment for undergraduates.</a> Biochemistry and Molecular Biology Education. 37, 99-105 (2009).</li>
<li>Troelsen, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adult-type+hypolactasia+and+regulation+of+lactase+expression%5BTitle%5D)+AND+%22Biochimica+et+biophysica+acta%22%5BJournal%5D)">Adult-type hypolactasia and regulation of lactase expression.</a> Biochimica et biophysica acta. 1723, 19-32 (2005).</li>
<li>Juers, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+structural+view+of+the+action+of+Escherichia+coli+%28lacZ%29+beta-galactosidase%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">A structural view of the action of Escherichia coli (lacZ) beta-galactosidase.</a> Biochemistry. 40, 14781-14794 (2001).</li>
<li>Plimmer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+presence+of+lactase+in+the+intestines+of+animals+and+on+the+adaptation+of+the+intestine+to+lactose%5BTitle%5D)+AND+%22Journal+of+Physiology%22%5BJournal%5D)">On the presence of lactase in the intestines of animals and on the adaptation of the intestine to lactose.</a> Journal of Physiology. 35, 20-31 (1906).</li>
<li>Krebs, H. A., Salvin, E., Johnson, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+formation+of+citric+and+alpha-ketoglutaric+acids+in+the+mammalian+body%5BTitle%5D)+AND+%22The+Biochemical+journal%22%5BJournal%5D)">The formation of citric and alpha-ketoglutaric acids in the mammalian body.</a> The Biochemical journal. 32, 113-117 (1938).</li>
<li>Vesa, T. H., Marteau, P., Korpela, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lactose+intolerance%5BTitle%5D)+AND+%22Journal+of+the+American+College+of+Nutrition%22%5BJournal%5D)">Lactose intolerance.</a> Journal of the American College of Nutrition. 19, 165S-175S (2000).</li>
<li>Robayo-Torres, C. C., Nichols, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+differentiation+of+congenital+lactase+deficiency+from+adult-type+hypolactasia%5BTitle%5D)+AND+%22Nutrition+Reviews%22%5BJournal%5D)">Molecular differentiation of congenital lactase deficiency from adult-type hypolactasia.</a> Nutrition Reviews. 65, 95-98 (2007).</li>
<li>Swallow, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetics+of+lactase+persistence+and+lactose+intolerance%5BTitle%5D)+AND+%22Annual+Review+of+Genetics%22%5BJournal%5D)">Genetics of lactase persistence and lactose intolerance.</a> Annual Review of Genetics. 37, 197-219 (2003).</li>
<li>Enattah, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Independent+introduction+of+two+lactase-persistence+alleles+into+human+populations+reflects+different+history+of+adaptation+to+milk+culture%5BTitle%5D)+AND+%22American+Journal+of+Human+Genetics%22%5BJournal%5D)">Independent introduction of two lactase-persistence alleles into human populations reflects different history of adaptation to milk culture.</a> American Journal of Human Genetics. 82, 57-72 (2008).</li>
<li>Lowe, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+rapid+detection+of+lactose+fermentation+in+paracolon+organisms+by+the+demonstration+of+beta-D-galactosidase%5BTitle%5D)+AND+%22Journal+of+Medical+Laboratory+Technology%22%5BJournal%5D)">The rapid detection of lactose fermentation in paracolon organisms by the demonstration of beta-D-galactosidase.</a> Journal of Medical Laboratory Technology. 19, 21-25 (1962).</li>
<li>Russo, S. F., Moothart, L. <a target="_blank" href="https://pubs.acs.org/doi/abs/10.1021/ed063p242">Kinetic study of the Enzyme Lactase.</a> Journal of Chemical Education. 63, 242(1986).</li>
</ol>

The authors have no conflicts of interest to disclose.

A detailed knowledge and understanding of enzymes, enzymatic reactions and enzyme kinetics is required for a wide range of topics spanning biology, biomedicine and pharmacology. The protocol described above uses lactase as an example of an enzyme relevant to human health for demonstrating enzymatic reactions, providing key skills that can be used in more advanced laboratory-based experiments.
This protocol has been deliberately designed to be as robust as possible, allowing students with little or no wet laboratory&#160;experience&#160;to produce interpretable results in a short space of time. In the 2014/2015 academic year at the University of Reading School of Pharmacy, a total of 144 students, organized into groups of 3, carried out the &#34;measuring lactase&#34; experiment, with all groups able to detect some level of enzymatic activity from lactase tablet extracts.
There are a number of critical steps in this protocol. First, it is essential that the initial extraction is efficient, allowing solubilization of enough active enzyme for subsequent analyses. In our experience, first year undergraduates can achieve this by following the protocol as outlined; however some guidance from laboratory demonstrators was required at this point. Although it would seem a self-evident point, a key factor in the success of this protocol is the ability to carefully measure volumes, correctly label tubes and follow instructions. While a certain level of proficiency can be assumed for many students, careful monitoring and a clear request for students to ask for assistance in the event that they are unsure of what to do next provides the greatest likelihood of a successful assay.
Student evaluation can be carried out by the in-class worksheet (available as supplemental material), asking students to note data from the experiments, comment on their results and link to background information/reading from lecture material, or by more detailed, out of class assessment with model data provided allowing for kinetic analyses to be attempted and evaluated.
The protocol presented herein is a simple example of a lactase activity assay under teaching laboratory conditions, and there is ample opportunity to increase the complexity of the experiment to allow more advanced analyses. In particular, the presented protocol provides an introduction to the concept of enzyme kinetics but does not allow for kinetic analysis of experimental data. As such, we would recommend viewing the described protocol as an initial template for classes, with the precise details adapted to suit the specific aims and talents of the student cohort undertaking the experiments. Examples of extended experiments could include: repeated measurements at different temperatures, using different substrate concentrations to allow statistical analysis, kinetic analysis of data (suitable for biochemistry students/majors), starting with several different tablets and allowing the students to evaluate different activity of enzyme in each (with the addition of control tablets, suitable for forensic science students/majors), assessing the impact of heat denaturation, or carrying out competition experiments by addition of lactose or inhibitors of lactase (Table 1). A detailed protocol for kinetic analysis of lactase has previously been published12.
An important strength of this protocol is that it combines a basic introduction to enzymology and enzyme kinetics with a real-life case study of enzymology in medicine. This makes this laboratory experiment of particular relevance to students of medicine, pharmacy, biomedical sciences and related subjects. Importantly, the fact that there is both a serious medical condition and a more widespread dietary issue associated with lactose catabolism provides the opportunity to raise a wide range of medical and ethical issues with the students. This could include the issues surrounding genetic testing for medical conditions, and associated discussions relating to gene therapy and genetic counselling, as well as the use and sale of medicinal supplements. One major limitation is that the protocol does not allow for precise estimation of enzyme concentration, due to the starting material used for the extraction. A modification to account for this, however, would be to use reagent grade enzyme for the assay. Importantly, this would also allow for more precise calculation of kinetics for the lactase enzyme.
In conclusion, assaying the activity of lactase in a teaching lab environment provides a robust, engaging and interesting introduction to the field of enzyme biology for early stage university students.

Enzymes are a specialized type of protein that act as biological catalysts for chemical reactions in living organisms1. The action of enzymes is critical for life, providing energy, disposing of waste and allowing organisms to function. Understanding enzymes is, therefore, critical for a full understanding of life. Such knowledge is essential for a wide variety of university level degree programs, ranging from the medical sciences to biology. While a detailed background ranging from principles of catalysis to theoretical models of enzyme activity can be provided to students using lectures and reading material, the properties of enzyme reactions are best comprehended by hands on practical experience of enzymes in action as previously demonstrated2. This protocol provides a simple to follow experimental paradigm for measuring enzyme activity under laboratory conditions, using lactase as an example of an enzyme with an activity relevant to human nutrition and health.
The glycosidic hydrolase lactase (EC 3.2.1.23/26) is an enzyme of central nutritional importance for mammals3. The activity of lactase is highly conserved through evolution, and derives from the beta-galactosidase family of enzymes &#8212; a family present from Escherichia coli through to Homo sapiens (Figure 1, PDB 1JZ8)4. The critical importance of lactase in a human nutritional setting stems from its role in allowing the breakdown of lactose into its constituent monosaccharide components, which can then be used to generate energy in the body. Lactase catalyzes the hydrolysis of the glycosidic bond in the disaccharide lactose, releasing galactose and glucose (Figure 2)5. These monosaccharides are then used primarily for the generation of adenosine triphosphate (ATP) via the citric acid cycle and oxidative phosphorylation6. During neonate and infant development, lactase is highly expressed in the human digestive system, breaking down lactose received from breast milk of which lactose is the primary carbohydrate component, and one of the key sources of nutrition during early years7. The medical importance of lactase is highlighted by congenital lactase deficiency (CLD), a rare autosomal recessive condition caused by mutations in the lactase gene (LCT) coding for the lactase enzyme8. New-born babies with CLD exhibit very little lactase activity, thus they cannot be fed on breast milk, any other type of milk, or formula containing lactose.
During childhood, lactase expression is normally reduced; however, this reduction following weaning varies geographically, with approximately 35% of adults worldwide continuing to express the enzyme9. Sustained expression of lactase, known as lactase persistence, allows individuals to continue to digest milk and dairy products from a range of sources. Conversely, the loss of lactase expression can lead to lactose intolerance, also known as adult-type hypolactasia (ATH), resulting from an inability to break down lactose in the gut. ATH is characterized by a build up of lactose in the colon following ingestion of lactose containing food products. In the colon, the accumulated lactose is fermented by gut microbial fauna, releasing gasses including hydrogen, methane, and carbon dioxide. The production of these gases in individuals with lactase enzyme deficiencies promote abdominal bloating, increased flatulence, pain, nausea, and borborygmi (stomach rumbling)7. Increased levels of lactose in the digestive tract can also lead to loose stools.
The control of LCT gene expression is modulated by polymorphisms located in introns of the nearby MCM6 gene. Individuals with sustained expression of lactase carry polymorphisms that function as strong distal enhancers for LCT gene expression, thus compensating the normal down regulation of LCT transcription during weaning, and consequently sustaining lactase expression in adulthood3. Enhancer polymorphisms have been suggested to have been positively selected following the domestication of cattle and camels in the Middle East over five thousand years ago9,10.
The symptoms resulting from ATH can be managed by reducing lactose intake, for example by removing dairy products from the diet. An alternative approach for ATH, and the approach of choice for CLD, is the use of lactase supplements, widely available from pharmacies. These supplements provide lactase isolated from a variety of sources, including yeast and bacteria, in a liquid or pill-based form that can be taken with or added to lactose containing food. The supplement will hydrolyze a proportion of the lactose present in the food to glucose and galactose products, thus permitting their absorption and preventing accumulation of undigested lactose substrate in the gut.
Based upon the use of lactase supplements as a dietary aid, we have developed a simple enzymology laboratory experiment suitable for first year biomedical science or pharmacy students. This laboratory experiment takes advantage of commercially available lactase supplements, and uses ortho-nitrophenol-beta-D-galactopyranoside (ONPG) to provide a colorimetric end point for measuring cleavage of glycosidic bonds by lactase (Figure 3)11. ONPG acts an artificial substrate for lactase, which when subjected to hydrolysis by this enzyme produces D-galactose and ortho-nitrophenol. The latter product has a yellow color, absorbing light at a wavelength of 420 nm. By quantifying any changes in absorbance at 420 nm following the exposure of ONPG to lactase, it is possible to estimate the activity of this enzyme. This laboratory experiment provides a demonstration of enzymatic hydrolase activity. By building in additional replicates and carrying out assays in different conditions, it is possible to incorporate more sophisticated analyses of enzyme kinetics, providing a valuable real-life example of enzymes in action relevant to human health.

<table><tbody><tr><td>Lactase tablet</td><td>Lamberts</td><td>8511-60</td><td></td></tr><tr><td>Phosphate buffered saline (powdered)</td><td>Sigma</td><td>P3813</td><td>Dissolved in deionized water to a final concentration of 100 mM</td></tr><tr><td>ONPG</td><td>Sigma</td><td>N1127</td><td>Dissolved in deionized water to a final concentration of 5 mM</td></tr><tr><td>Sodium Carbonate</td><td>Sigma</td><td>451614</td><td>Dissolved in deionized water to a final concentration of 1 M</td></tr><tr><td>De-ionized water</td><td>NA</td><td>NA</td><td></td></tr><tr><td>Pestle and mortar</td><td>VWR</td><td></td><td></td></tr><tr><td>Spectrophotometer</td><td>Jenway 6315</td><td></td><td></td></tr><tr><td>Pipettes</td><td>Gilson</td><td></td><td></td></tr><tr><td>15 mL tubes</td><td>VWR</td><td></td><td></td></tr><tr><td>1.5 mL tubes</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Spectrophotometer cuvettes</td><td>Jenway</td><td></td><td></td></tr><tr><td>Vortex</td><td>Vortex genie 2</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Beckman</td><td></td><td></td></tr><tr><td>Ice bucket</td><td>VWR</td><td></td><td></td></tr><tr><td>Water bath</td><td>Thermo-Scientific</td><td></td><td></td></tr><tr><td>Weighing scales</td><td>Thermo-Scientific</td><td></td><td></td></tr></tbody></table>

measuring lactase enzymatic activity in the teaching lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

Representative results for Section 2 of the protocol are shown in Figure 4A. The control reaction, in the absence of lactase enzyme, remains clear whilst the reaction solution, containing extract from the lactase tablet, turns yellow as ONPG is hydrolyzed releasing ortho-nitrophenol. Figure 4B shows quantification using arbitrary units from Section 4 of the protocol, following analysis of samples using the spectrophotometer. Production of ortho-nitrophenol is lowest when the reaction is incubated on ice, and highest when the reaction is carried out at 37 °C.
<img alt="Protein structure diagram, molecular interaction visualization, highlighting binding site details." class="xfigimg" src="/files/ftp_upload/54377/54377fig1.jpg" data-altupdatedat="1747929244000" data-alt="Figure 1"> 
Figure 1: E. coli beta-galactosidase. (A) Crystal structure of beta-galactosidase from Escherichia coli, showing the tetrameric structure of the active complex. (B) Allolactose (shown in red) bound to the active site of beta-galactosidase. Images generated from PDB 1JZ84. <a href="https://www.jove.com/files/ftp_upload/54377/54377fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Lactose hydrolysis equation with lactase enzyme; diagram of lactose to D-galactose and D-glucose." class="xfigimg" src="/files/ftp_upload/54377/54377fig2.jpg" data-altupdatedat="1747929244000" data-alt="Figure 2"> 
Figure 2: Enzymatic action of lactase. Hydrolysis of lactose by lactase to produce beta-D-galactose and beta-D-glucose. <a href="https://www.jove.com/files/ftp_upload/54377/54377fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="ONPG hydrolysis chemical equation, lactase enzyme reaction diagram, D-galactose, ortho-nitrophenol." class="xfigimg" src="/files/ftp_upload/54377/54377fig3.jpg" data-altupdatedat="1747929244000" data-alt="Figure 3"> 
Figure 3: Hydrolysis of ortho-nitrophenol-beta-D-galactopyranoside. ONPG hydrolysis by lactase to produce beta-D-galactose and ortho-nitrophenol (which is yellow in color), allowing estimation of lactase activity by measurement of absorbance at 420 nm. <a href="https://www.jove.com/files/ftp_upload/54377/54377fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cuvette absorption experiment; diagram showing control vs reaction absorption at varied temperatures." class="xfigimg" src="/files/ftp_upload/54377/54377fig4.jpg" data-altupdatedat="1747929244000" data-alt="Figure 4"> 
Figure 4: ONPG hydrolysis. Representative results of ONPG hydrolysis, showing control and reaction tubes (A) and representative data from Section 4 of the protocol recorded on in-class worksheets, showing raw absorbance in arbitrary units at 420 nm, corrected for background and at three different temperatures (B). <a href="https://www.jove.com/files/ftp_upload/54377/54377fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Experimental Condition</td>
			<td>Experimental variables</td>
		</tr>
		<tr>
			<td>Temperature</td>
			<td>0 °C, 10 °C, 20 °C, 30 °C, 50 °C</td>
		</tr>
		<tr>
			<td>Substrate concentration</td>
			<td>0 mM, 1 mM, 5 mM ,10 mM ONPG</td>
		</tr>
		<tr>
			<td>Competitive inhibition</td>
			<td>5 mM ONPG plus 0 mM, 1 mM, 5 mM, or 10 mM lactose</td>
		</tr>
		<tr>
			<td>Time dependence</td>
			<td>0 s, 15 s, 30 s, 1 min, 5 min, 10 min, 20 min</td>
		</tr>
		<tr>
			<td>Heat denaturation</td>
			<td>Lactase extract heated to 100 °C for 10 min prior to assay</td>
		</tr>
	</tbody>
</table>
Table 1: Potential extended experimental condition. Suggested extended experiments, to be carried out in triplicate, investigating specific aspects of lactase biology.
Supplemental Materials. <a href="https://www.jove.com/files/ftp_upload/54377/54377_SupplementalFiles.pdf" target="_blank">Please click here to download this file.</a>

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Measuring Lactase Enzymatic Activity in the Teaching Lab

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...

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113.4K Views.  University of Reading. The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

Video: Measuring Lactase Enzymatic Activity in the Teaching Lab

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.

We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

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The thermal stability of enzyme activity is readily measured by isothermal titration calorimetry (ITC). Most protein stability assays currently used measure protein unfolding, but do not provide information about enzymatic activity. ITC enables direct determination of the effect of enzyme modifications on the stability of enzyme activity.

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Environment

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Bioengineering

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

The pancreatic acinar cell is the main parenchymal cell of the exocrine pancreas and plays a primary role in the secretion of pancreatic enzymes into the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro. These techniques provide a powerful tool to characterize acinar cell physiology and pathology.

We describe a reproducible method of preparing mouse pancreatic acinar cells from a mouse for the purpose of examining acinar cell calcium signals and cellular injury with physiologically and pathologically relevant stimuli. A method for adenoviral infection of these cells is also provided.

The pancreatic acinar  cell is the main parenchymal cell of the exocrine pancreas and plays a primary  role in the secretion of pancreatic enzymes into ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Enzyme Activity

<h4>Biological Catalysts</h4>

All living organisms continuously perform numerous biochemical reactions to sustain their presence. Most of these reactions require an input of energy to start, which is called the activation energy. Catalysts are chemicals that lower the activation energy. Even though catalysts facilitate a chemical reaction, they are not consumed by it. This means a catalyst can facilitate a specific chemical reaction over and over in succession, increasing the rate of that reaction.

Enzymes are biological catalysts that increase the speed and efficiency of biochemical reactions. Most enzymes are proteins but certain ribonucleic acid molecules also have catalytic properties. Each type of enzyme can only bind to one or a few specific substrates, therefore, there is a vast diversity of enzymes, all with distinct functions. The specific reaction of an enzyme depends on its active sites, which has shapes that closely matches the shape of its substrates. Upon substrate binding, the enzyme changes shape slightly in a way that allows the chemical reaction to happen more readily, thus reducing the activation energy of the reaction. Once the reaction takes place, the enzyme releases the products and returns to its original shape, which allows it to repeat the process again.

<h4>Catabolytic versus Anabolic Enzymes</h4>

Most enzymatic reactions can be categorized as either catabolic and anabolic, which have opposite functions. Enzymes that mediate catabolic reactions break down larger substances into multiple products. A well-known catabolic enzyme is lactase, which splits the disaccharide lactose in dairy products into monosaccharides galactose and glucose. Humans have large amounts of lactase in their small intestine after birth, however many individuals lose more than 90% of their lactase by early childhood1. This reduction diminishes the ability to digest lactose in an age-dependent manner, which is known as lactose intolerance2.

Anabolic enzymes combine multiple substances into a single product. For example, the enzyme DNA polymerase synthesizes DNA molecules by joining nucleotides together. Similarly, the enzyme glycogen synthase connects glucose molecules to generate glycogen. Insulin regulates the activity of glycogen synthase, therefore diabetic patients with low to no insulin levels cannot synthesize glycogen without insulin treatment3.

<h4>Environmental Factors</h4>

The shape and charge of the active site of an enzyme is tailored to the specific chemical reaction it catalyzes, therefore the enzyme activity is influenced by factors that affect its active site. Hence, enzymes require specific conditions to work at optimal levels and depending on their function and location, these conditions can vary greatly. These factors include pH and temperature. For example, active sites of enzymes often contain acidic or basic amino acids. Deviations from the optimum pH alter the charges in the active site, making it difficult for the substrates to interact with the enzyme. This property has been used for centuries to preserve food by pickling, which reduces the pH of the pickled food item to a level that effectively inhibits microbial enzymatic activity. Similarly, each enzyme works at an optimal temperature range. If the temperature shifts below or above this range, it alters the shape of the active site, preventing it from effectively interacting with its substrates. In most cases, returning to the optimal temperature restores the enzyme&#8217;s original shape and function, however, temperatures that are too high can denature or irreversibly damage the enzyme structure. Again, these properties are useful for food safety; we cook meat thoroughly to kill harmful bacteria by denaturing their enzymes and refrigerate most food items to slow down enzymatic activity of microorganisms.

Extremophiles, or organisms found in extreme conditions that are lethal to most forms of life, have enzymes capable of functioning at those extremes. For instance, Thermus aquaticus is a bacterium found in hot springs and hydrothermal vents reaching temperatures at which most proteins would denature. Taq polymerase is a thermostable DNA polymerase found in T. aquaticus, which has allowed the development of polymerase chain reaction (PCR), a widely-used laboratory method for rapid DNA replication. PCR requires high temperatures to separate the strands of the DNA double helix to be replicated. Taq polymerase remains stable at these high temperatures while most enzymes would be degraded. PCR cycles between high temperatures that break the DNA into single strands and lower temperatures that allow the polymerase to build new double stranded DNA.

<h4>Molecular Factors that Influence Enzyme Activity</h4>

In addition to specific pH and temperature ranges, some enzymes function better in the presence of other molecules or ions. Coenzymes are biomolecules that bind to the active site of the enzyme and participate in catalysis without getting consumed by the reaction. Coenzymes often transport electrons, specific atoms and functional groups, and thus act as intermediary carriers. B vitamins act as coenzymes for numerous biochemical reactions. For instance, coenzyme A (coA), which is the active form of pantothenic acid, is required by 4% of all mammalian enzymes4. Cofactors are often classified as inorganic substances, such as ions, that are required for the enzymatic function. For example, all DNA polymerase enzymes require Mg2+ or Mn2+ for DNA replication as well as excision of incorrectly incorporated nucleotides5.

There are also other molecules that are not necessary for an enzyme to function but are capable of altering the enzymatic activity. Substances that increase the enzyme activity are called activators, whereas others that decrease the enzyme activity are called inhibitors. These molecules bind to enzymes either at the active site or other locations on the enzyme known as allosteric sites. Sometimes these molecules can bind to the exact location in the active site as the substrate, and thus compete with the substrate for the enzyme binding. Others, especially the substances that bind to allosteric sites, can alter enzyme function without blocking the substrate binding to the active site, and are thus non-competitive. Moreover, binding of inhibitors can be reversible or irreversible. When the interaction is reversable, the enzyme can resume its function once the inhibitor detaches. However, when the binding is irreversible, the inhibitor never detaches from the enzyme and the enzyme becomes irreparably damaged and needs to be replaced. Nevertheless, enzyme inhibitors are useful as well. For instance, inhibitors have been utilized as antibiotics to inhibit bacterial growth, and as chemotherapeutics to inhibit the uncontrolled division of cancer cells6.

<h4>Measuring Enzyme Function</h4>

One method used by scientists to study the effects of different conditions on enzyme function is to quantify the reaction rate, which is how fast the enzyme is catalyzing the reaction. Therefore, a higher reaction rate indicates that the enzyme is catalyzing reactions more efficiently, whereas a lower rate means that it is working less efficiently. To determine the reaction rate, the reaction velocity is calculated by measuring the amount of product made per unit time. This is done by measuring the product concentration starting right at the beginning of the reaction when the product concentration is increasing linearly, until the enzyme is completely saturated with the substrate and the catalysis reaches a steady level. Once the baseline reaction rate is determined, it can be used as a reference point, or control, when comparing between a variety of treatments. We can assess reaction rates as relative or absolute measures. A relative reaction rate is a comparative measure that shows differences between two or more conditions, such as treatments in a single experiment, but does not give a value for what is being measured. An absolute reaction rate relies on a quantitative measure of the progress of the reaction. An easy way to understand the difference between relative and absolute reaction rates is to consider measuring the speed of a car in a race. A relative method would compare by assessing the ranking of cars at the finish line, whereas the absolute rate would assess and compare the speed of individual cars in miles per hour.

Since enzymes are so widely used, researchers have begun looking for ways to create and modify enzymes to suit specific needs, known as protein engineering. Recombinant DNA technology allows amino acid sequences to be changed in order to alter an enzyme&#8217;s shape or active site affinity. This has been used to create highly sensitive diagnostic tests for various conditions, as well as for bioremediation efforts and waste management tools7.

<h4>References</h4>

<ol class="references">
	<li>Swagerty DL Jr, Walling AD, Klein RM. Lactose intolerance. Am Fam Physician. 2002, Vol. 65, 9: 1845-50.</li>
	<li>Lomer MC, Parkes GC, Sanderson JD. Review article: lactose intolerance in clinical practice--myths and realities. Aliment Pharmacol Ther. 2008, Vol. 27, 2: 93-103.</li>
	<li>AR, Saltiel. New perspectives into the molecular pathogenesis and treatment of type 2 diabetes. Cell. 2001, Vol. 104, 4: 517-29.</li>
	<li>DO, Kennedy. B Vitamins and the Brain: Mechanisms, Dose and Efficacy--A Review. Nutrients. 2016, Vol. 8, 2: 68 doi:10.3390/nu8020068.</li>
	<li>Vashishtha AK, Wang J, Konigsberg WH. Different Divalent Cations Alter the Kinetics and Fidelity of DNA Polymerases. J Biol Chem. 2016, Vol. 291, 40: 20869-20875.</li>
	<li>Watson ID, Stewart MJ, Platt DJ. Clinical pharmacokinetics of enzyme inhibitors in antimicrobial chemotherapy. Clin Pharmacokinet. 1988, Vol. 15, 3: 133-64.</li>
	<li>Ahuja SK, Ferreira, GM, Moreira AR. Utilization of Enzymes for Environmental Applications. Crit Rev Biotech. 2004, Vols. 2-3, 125-54.</li>
</ol>

Enzyme Activity: Effect of pH and Temperature on Peroxidase Activity - Concept

Biological Catalysts

All living organisms continuously perform numerous biochemical reactions to sustain their presence. Most of these reactions require ...

JoVE Lab Manual

Cellular Processes

Evaluating Cytotoxicity and Membrane Integrity in Lung Slices via Lactate Dehydrogenase Assay

Source: Neuhaus, V., et al. <a href="https://app.jove.com/v/57042">Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices.</a> J. Vis. Exp. (2018).
This video demonstrates the lactate dehydrogenase, LDH assay to measure the cytotoxic effect of a test substance and its effect on the membrane integrity in human precision-cut lung slices. The assay provides a quantitative measure of cell death or damage in the sample.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines
1. General Preparation of Human PCLS ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

Quantifying Bulk Autophagic Sequestration Activity in Mammalian Cells

Source: Luhr, M. et al., <a href="https://app.jove.com/v/57971">The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells.</a> J. Vis. Exp. (2018)
The video demonstrates an assay for assessing bulk autophagic sequestration activity in mammalian cells. It utilizes electroporation to release cytosolic proteins, including lactate dehydrogenase (LDH). The sequestered LDH and total cellular LDH fractions are determined to assess LDH sequestration, providing insights into the overall bulk sequestration activity.

1. Cell Seeding and Treatment

	Culture adherent cells in 75 cm2&#160;tissue culture flasks in a humidified incubator with 5% CO2&#160;at 37 &#176;C, ...

Cellular Immunodiagnostics

Colorimetric Assay for Quantification of Lactate in Test Samples

To measure the concentration of lactate, use a colorimetric assay kit to carry out duplicate analysis for the test samples. Add 10 microliters of the test samples to each well of a 96-well plate, and then add lactate assay buffer to each sample to adjust the volume to 50 microliters.
Dilute 100 millimolar L(+)-Lactate standard with lactate assay buffer to 1 millimolar. Then, into a series of wells, add 0, 2, 4, 6, 8, and 10 microliters of diluted L(+)-Lactate standard. Add 50 microliters of reaction mix into each well, and incubate at room temperature protected from light for at least 30 minutes for the color to develop.
Finally, use a microplate reader to measure the absorbance of each well at 570 nanometers. Subtract the absorbance of the background control mix from the absorbance of the reaction mix. Then, plot the lactate standard curve, and calculate the lactate concentrations from the curve by multiplying each concentration by 2.25.

Measuring Lactase Enzymatic Activity in the Teaching Lab

Summary

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An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

The Lactate Dehydrogenase Sequestration Assay — A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Enzyme Activity

Evaluating Cytotoxicity and Membrane Integrity in Lung Slices via Lactate Dehydrogenase Assay

Quantifying Bulk Autophagic Sequestration Activity in Mammalian Cells

Colorimetric Assay for Quantification of Lactate in Test Samples