Name: determining the serum stability of human adenosine deaminase 1 enzyme
Uploaded: 2024-09-27T14:25:00.000+00:00
Duration: 4 min 17 s
Description: The concept of enzyme stability is typically used to refer to an enzyme's thermostability - its ability to retain structure and activity as temperature ...

This work was supported by the National Institutes of Health [1DP2CA280622-01] and funding from Biolocity. We thank Dr. Maria Jennings and Andrea Fox for providing the HsADA1 and HsKYNase expression vectors.

1. Serum incubation
<ol>
	<li>Prepare a 10x stock of HsADA1 in 1x PBS pH 7.4 (1x PBS) at a final concentration of 10 &#181;M. Thaw a 15 mL aliquot of pooled human serum and pre-warm it to 37 &#176;C. Prewarm a 50 mL aliquot of 1x PBS to 37 &#176;C.</li>
	<li>Prepare the enzyme-serum incubation mixtures by adding 100 &#181;L of the 10x HsADA1 stock to 900 &#181;L of pooled human serum in a low-bind microcentrifuge tube, referred to as the enzyme + serum mixture. In a separate low-bind microcentrifuge tube, add 100 &#181;L of the 10x HsADA1 stock to 900 &#181;L of 1x PBS for comparison to a non-serum control, referred to as the enzyme + 1x PBS mixture. The final concentration of HsADA1 in each mixture will be 1 &#181;M.</li>
	<li>Prepare an additional control in a low-bind microcentrifuge tube consisting of 100 &#181;L of 1x PBS mixed with 900 &#181;L of pooled human serum. This will serve as the non-enzyme control for the enzyme + serum mixture. Additionally, make a control consisting of 1 mL of 1x PBS. This will serve as the non-enzyme control for the enzyme + 1x PBS mixture.</li>
	<li>Aliquot 100 &#181;L of each mixture from step 1.2 into separate low-bind tubes and dilute 1:1 with 100 &#181;L of 30% (v/v) glycerol in 1x PBS. Flash freeze with liquid nitrogen and store at -80 &#176;C. These samples will be the Day 0 timepoint, and the concentration of HsADA1 in them will be 500 nM. Repeat this step for the non-enzyme control mixtures from 1.3. Seal all enzymes and control mixtures with transparent film and place them into a 37 &#176;C incubator.</li>
	<li>After 24 h, aliquot 100 &#181;L of each incubated sample and dilute 1:1 with 100 &#181;L of 30% (v/v) glycerol in 1x PBS. Flash freeze with liquid nitrogen and store at -80 &#176;C. These samples will be the Day 1 timepoint. Return all enzyme and control mixtures to the 37 &#176;C incubator. Repeat this step at 72 h and 120 h. 
	NOTE: Aliquoted samples do not necessarily need to be flash-frozen. Alternatively, the user may perform the microplate-based assay immediately after taking each sample, although fresh standard curves and adenosine substrate stocks should be prepared each time a sample is tested.</li>
</ol>
2. Microplate-based assay
<ol>
	<li>Prepare the microplate reader by setting up a kinetic read protocol (Figure 2) to measure the absorbance at 260 nm with the shortest reading interval for 30 min and preheat the reader to a setpoint of 37 &#176;C.</li>
	<li>Thaw all collected samples on ice. Dilute each thawed sample 1:10 using 1x PBS prewarmed to 37 &#176;C as a diluent in low-bind microcentrifuge tubes. The new HsADA1 concentration in these samples will be 50 nM.</li>
	<li>Make a 10x dilution into 1x PBS from the non-enzyme serum control sample as well. Additionally, prepare a 5 mM adenosine stock by adding 66.8 mg of adenosine to 50 mL of 1x PBS. Prepare a 250 &#181;M dilution of adenosine by adding 400 &#181;L of the adenosine stock to 7,600 &#181;L of 1x PBS and prewarm it to 37 &#176;C in an incubator.</li>
	<li>To a 96-well UV-compatible microplate, add 160 &#181;L of the 250 &#181;M adenosine dilution and 40 &#181;L of each diluted protein sample in triplicate for a total volume of 200 &#181;L. This will yield a final adenosine concentration of 200 &#181;M and a final enzyme concentration of 10 nM.</li>
	<li>Repeat the dilution for the control samples by adding 40 &#181;L of the diluted serum or 1x PBS controls and 160 &#181;L of the 250 &#181;M adenosine dilution in triplicate. This will also yield a final adenosine concentration of 200 &#181;M but without enzyme. 
	NOTE: It may be beneficial to add all diluted enzyme and control samples individually to the microplate first and then add the adenosine substrate with a 12-channel micropipette to reduce the total loading time. Additionally, UV-transparent plates must be used in this step to prevent the plate itself from confounding the measured absorbance signal.</li>
	<li>Measure the absorbance of each well at 260 nm for 30 min at 37 &#176;C. Once done, export the data to a spreadsheet for further analysis.</li>
</ol>
3. Analysis of microplate reader data
<ol>
	<li>Determine the slope of the linear region of the absorbance data as a function of time for the first several minutes of the microplate data. For the serum-diluted samples, this corresponded to the first ~120 s. For the 1x PBS-diluted samples, this corresponded to the first ~270 s. This slope of declining absorbance values will have a negative numerical value and will have units of change in absorbance units per second (&#916;A260/s).</li>
	<li>Subtract out the slope of the negative control consisting of pooled human serum from the slope of the enzyme + serum data. Perform the same operation on the enzyme + 1x PBS mixtures by subtracting out the slope of the 1x PBS control.</li>
	<li>Normalize all the adjusted slopes to the original slope at t=0 h to obtain the fraction of activity remaining using Equation 1. 
	<img alt="Equation 1" src="/files/ftp_upload/67216/67216eq01.jpg" style="margin: auto;" />&#160; &#160; Equation 1</li>
	<li>Plot the fraction of activity remaining versus time.</li>
</ol>

Evaluating Serum Stability of HsADA1: A Microplate-Based Approach

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JB and MRJ are inventors on patents or patent applications related to adenosine deaminase and/or kynureninase enzymes. All other authors have no conflicts of interest to disclose.

This protocol uses absorbance change as the substrate is converted to the product to gauge the activity of an enzyme. As such, the substrate and product must have distinct spectral profiles. This is the case with adenosine and inosine both having distinct spectral profiles and extinction coefficients between 260-265 nm6,8,12,13. This assay is inspired by several previous works. Kalackar, for example, utilized the change in absorbance at 265 nm as a direct correlate for adenosine deaminase activity14. Gracia et al. and Lu et al. employed an approach rooted in the Beer-Lambert law, which relates the initial velocity to the difference in the extinction coefficients between adenosine and inosine8,13. In a similar vein, OH-Kyn and OH-AA also have distinct spectral profiles in that OH-Kyn absorbs strongly at 379 nm while OH-AA does not9,10. The absorbance spectrum of metabolites can be easily checked using a full-spectrum scan on many microplate readers, which would, in turn, inform the user whether it is possible to track the progress of an enzymatic reaction. As stated earlier, Lu et al. have previously analyzed native HsADA1 stability in different bodily fluids, and Jennings et al. have employed a very similar method to gauge the serum stability of recombinant HsADA1 and a mutant enzyme6,8. This method, like that of Jennings et al., is unique in that we can use recombinantly expressed enzymes as opposed to natively derived ones. In doing so, this method can be extrapolated to mutant proteins not natively found within living organisms.
For product/substrate combinations that cannot be measured using a plate-based absorbance assay, this protocol can be modified to incorporate several other alternative analytical methods that track either substrate degradation or product formation for kinetic assays, such as high-performance liquid chromatography (HPLC), which for instance, has previously been used to assess the kinetics of both adenosine deaminase and kynureninase15,16,17. Only the method to determine the remaining substrate or created product after a certain time (if not determined continuously) should vary. The choice of the assay will, of course, be dependent on the reaction being catalyzed by the enzyme of interest, and a key consideration in this choice will also be the compatibility of a given analytical method with diluted human serum. Of equal importance may also be the total time required to analyze all samples of interest, and a key benefit of utilizing the microplate assay is the ability to easily analyze degradation rates in up to 96 samples at a time.
Assay parameters such as the on-plate enzyme and substrate concentrations will need to be tuned for individual enzymes of interest. For all the assays conducted here, a substrate concentration above Km by an order of magnitude greater (1.15-1.26 OOM) was chosen. Wildtype HsADA1 has a Km of ~14 &#181;M for adenosine, and the final on-plate adenosine concentration used was 200 &#181;M3. Similarly, the engineered HsKYNase variant tested has a Km ~22 &#181;M for OH-Kyn, and the final on-plate OH-Kyn concentration used was 400 &#181;M7. One important requirement for the substrate concentrations of interest for the microplate assay is that the substrate obeys the Beer-Lambert law within the given range, such that an absorbance decrease can be linearly correlated with substrate degradation (Supplementary Figure 1 and Supplementary Figure 2). This linear correlation removes the need to convert absorbance change to substrate concentration, as performing this conversion would not change the remaining activity fraction.
As for the enzyme concentration, the main criterion to consider is for there to be sufficient substrate degradation relative to the background. For both HsADA1 and the engineered HsKYNase, we used previously published literature values to generate representative results6,7. As we have shown here, using shorter times with higher enzyme concentrations can be feasible, as can using longer times and lower enzyme concentrations. In general, a longer time period/lower enzyme concentration may give more reproducible results unless the substrate itself is prone to degradation. In this vein, both kynurenine and its derivatives, including OH-Kyn, degrade relatively quickly at room temperature, resulting in a noticeable yellowing of the substrate mixture18. For other enzymes, a simple concentration escalation experiment can be conducted to identify concentrations with the most optimal reaction curves and background separation. Performing this concentration escalation on HsADA1 shows that within a 1-10 nM range, the greatest background separation is achieved (Supplementary Figure 3). Performing this same concentration escalation experiment with engineered HsKYNase shows a similar trend with the greatest background separation at 2 &#181;M (Supplementary Figure 4). However, both assays (HsADA1 versus HsKYNase) do not possess the same sensitivity. For HsADA1, an on-plate enzyme concentration one-tenth of that used in the protocol is still distinguishable from background at statistically significant levels (Supplementary Figure 3). The same does not hold true for HsKYNase, meaning it may be harder to quantitatively assess how much activity HsKYNase retains once it has lost more than 90% of its activity. This represents a potential limitation of the microplate reader method.
Additionally, one observation we made for HsKYNase was that upon plate loading, there was an immediate gap between the absorbance readings between the enzyme + 1x PBS samples and the 1x PBS control (Figure 5B,D). This gap was present for all the time points. Because we also observed this phenomenon when testing heat-deactivated enzyme (heated at 95 &#176;C for 10 min), we concluded that it was not a result of enzyme-mediated substrate degradation (Supplementary Figure 5). While we proceeded with original control absorbance values, future studies could use deactivated enzyme controls to eliminate this discrepancy. However, the cost to purchase or produce the enzyme of interest should be taken into account. For both HsKYNase and HsADA1, there was some variability in the starting absorbance values of the test samples, with some starting at a lower absorbance value than their associated controls (Figure 3 and Figure 5). This could be partially attributed to the lag time in adding substrate to all the protein samples, followed by loading into the plate reader. However, because the substrate concentration used is much higher than the Km values of the used enzymes, this is not expected to lead to substrate concentration-dependent effects on the observed slopes.
In the protocol and representative results section, samples are aliquoted at the indicated time points, with the sampling time varying based on the activity profile of an enzyme. This sampling schedule will depend on the activity profile of an enzyme of interest in serum. If an enzyme loses activity quickly, then a smaller sampling interval will be desired, as is the case with the HsKYNase_66-W102H-T333N mutant tested, and the opposite is true for enzymes with greater serum stability. As such, it is likely beneficial to run a trial of this protocol whereby aliquots are sampled in both small intervals (1 h, 2 h, etc.) and large ones (24 h, 48 h, etc.) to survey what the serum stability profile looks like for a new enzyme of interest. Of course, as it becomes more robust, the serum stability will become less relevant compared to renal clearance or other mechanisms of in vivo protein clearance.
Because kynureninases differ from HsADA1, we modified several assay parameters, namely enzyme concentration during incubation in serum or 1x PBS, sampling interval, microplate assay enzyme concentration, microplate assay substrate concentration, and, of course, the substrate itself. The changes are: the 10x enzyme stock was at a concentration of 100 &#181;M, which, after diluting with serum/1x PBS, resulted in a concentration of 10 &#181;M. The sampling interval in the serum incubation method was adjusted because the HsKYNase loses activity faster in serum than in HsADA1. Therefore, the method included sampling at 0.5 h, 3 h, and 24 h time points. Additionally, HsKYNase samples were not flash frozen, instead being immediately analyzed using the microplate assay. The plate reader was configured to read at 379 nm to monitor the progress of the enzymatic reaction. Another important difference in the microplate-based assay when studying the HsKYNase enzyme is the preparation of the substrate. A 2 mM stock of OH-Kyn was made by dissolving 4.5 mg of OH-Kyn in 10 mL of 1x PBS. At the same time, 6 mg of pyridoxal phosphate (PLP), the HsKYNase co-factor, was dissolved into 1 mL of 1x PBS to make a 100x stock of PLP. A 500 &#181;M dilution of OH-Kyn was prepared by adding 2.5 mL of OH-Kyn stock to 7.5 mL of 1x PBS as well as 100 &#181;L of the 100x PLP stock. To run the microplate assay, 80 &#181;L of the 500 &#181;M OH-Kyn dilution and 20 &#181;L of the aliquoted protein samples or negative controls were added in triplicate to a 96-well UV-compatible microplate. This yielded a final OH-Kyn concentration of 400 &#181;M and a final enzyme concentration of 2 &#181;M, after which the absorbance at 379 nm was measured for 30 min at 37 &#176;C. Unlike HsADA1, which was diluted before being analyzed on the plate reader, HsKYNase was not diluted.
While this protocol is relatively facile, a potential challenge could lie in preparing the original enzyme stocks. All the enzymes used in the representative results section were expressed in E. coli and purified using nickel-based affinity chromatography followed by size exclusion chromatography (SEC). Several other sources have expertly described alternatives to our method of purification, spanning host organisms and downstream processing19,20. This assay fundamentally tries to determine the ability of an enzyme to retain its activity in pooled human serum. One assumption in our methodology is that an enzyme&#39;s loss in activity is permanent, i.e., removing a deactivated enzyme from serum will not restore its activity. This is significant because, in this protocol, we dilute the enzyme mixtures further with glycerol and 1x PBS before flash freezing and after thawing. If this loss in activity is reversible upon removal of the serum, these additional dilutions will prevent the assay from capturing this phenomenon. If this is of concern to the user, then 1.) pooled human serum can be used as a diluent instead of 1x PBS in the preparation of all substrate and test sample solutions, and 2.) the flash freezing and addition of glycerol can be skipped, and samples can be immediately processed to maintain the enzyme in its original environment. Lastly, while this assay can be used to probe the serum-specific effects on enzyme activity, no conclusions can be made regarding the specific mechanism behind such effects. Whether this loss of activity is caused by enzyme unfolding, aggregation, loss of cofactor, or cleavage by serum proteases would need further investigation. Similar qualifications can be said of the 1x PBS incubated enzyme in that mechanistic conclusions cannot be derived from this assay.
In the context of developing protein biologics as therapeutics, the ability to retain activity in serum is an important metric21. A therapeutic enzyme that loses its activity very quickly would have to be dosed repeatedly to obtain clinical benefit. This work provides users with an in vitro assay to quantitatively screen enzymes for enhanced serum stability. If coupled with directed evolution efforts, enzymes with superior serum stability could be screened and selected for, giving greater potential for translatability. Additionally, we anticipate that this protocol can also work with more advanced systems, for example, to measure serum stability where the enzyme is encapsulated. Das et al. applied an assay for several different enzymes packaged in virus-like particles and tested their stability in different denaturing conditions, such as organic solvents22.

The following method allows a user to quantitatively assess an enzyme&#39;s ability to retain its activity when exposed to conditions that mimic what it will encounter following intravenous injection. The in vitro method mimics such in vivo conditions and consists of the incubation of the enzyme in pooled human serum at 37 &#176;C and time-coursed analyses of retention of enzyme activity. We refer to an enzyme&#39;s ability to retain activity in these conditions as its serum stability, and the analysis method for enzyme activity takes advantage of differences in absorbance between an enzyme&#39;s substrate and the resulting product. The concept of serum stability is not just enzyme-specific and has been applied to several other treatment modalities as well. For example, the serum stability of RNA aptamers targeting COVID spike proteins has previously been assessed by monitoring their degradation post-incubation with fetal calf serum1. Antibacterial peptides have also been assessed for their ability to suppress bacterial growth post-incubation with pooled human serum2.
HsADA1 is an enzyme that catalyzes the conversion of adenosine or 2-deoxyadenosine into inosine or 2-deoxyinosine, respectively3. Adenosine has an absorption peak of 260 nm, while inosine absorbs strongest at 250 nm4. This shift in absorption peaks can be detected on a microplate reader by a decrease in absorption intensity at 260 nm when HsADA1 is added to adenosine. The HsADA1 enzyme has important implications in the human body, and its deficiency causes a severe combined immunodeficiency (ADA-SCID)5. The bovine homolog of HsADA1, BtADA1, can be utilized as an enzyme replacement therapeutic for the treatment of ADA-SCID, and we have previously shown that wildtype HsADA1 loses its activity when incubated with pooled human serum, potentially hindering its use as a therapeutic6. Therefore, we selected the wildtype HsADA1 enzyme to demonstrate a procedure to determine an enzyme&#39;s serum stability. A detailed purification method for HsADA1 has been described previously6.
In the following protocol (as detailed in Figure 1), we demonstrate how to co-incubate wildtype HsADA1 in pooled human serum at 37&#160;&#176;C. During this time, test samples are taken at defined time points and flash-frozen for future analysis. Once all samples have been taken, a microplate assay is run whereby each sample is combined with the substrate, with the resulting absorbance decrease being a correlation for the retained activity of the enzyme. Representative results illustrating the serum stability of HsADA1 are shown, and because this metric may be relevant to determining the potential therapeutic value of other enzymes, we also discuss considerations to adapt this protocol to an engineered Homo sapiens kynureninase enzyme (HsKYNase) and any enzymes more generally. HsKYNase is an enzyme involved in the metabolism of tryptophan and is able to degrade the tryptophan-byproducts kynurenine and hydroxy-kynurenine (OH-Kyn) into anthranilic acid and hydroxy-anthranilic acid (OH-AA), respectively. Enzyme-mediated modulation of tryptophan metabolism may be of therapeutic relevance7.

<table><tbody><tr><td>Adenosine&amp;nbsp;</td><td>Sigma Aldrich</td><td>A9251-25G</td><td>25 g</td></tr><tr><td>BioTek Synergy HT Microplate Reader</td><td></td><td></td><td></td></tr><tr><td>Eppendorf LoBind Microcentrifuge Tubes: Protein&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>13-698-795</td><td>2 mL</td></tr><tr><td>Glycerol</td><td>Fisher Scientific&amp;nbsp;</td><td>G33-4</td><td>4 L</td></tr><tr><td>HsKYNase66-W102H-T333N</td><td>In-house</td><td></td><td></td></tr><tr><td>Human Serum, Pooled</td><td>MP Biomedicals</td><td>92931149</td><td>100 mL</td></tr><tr><td>Hydroxy-kynurenine</td><td>Cayman Chemicals</td><td>27778</td><td></td></tr><tr><td>Inosine&amp;nbsp;</td><td>TCI</td><td>I0037</td><td>25 g</td></tr><tr><td>PBS, 1x&amp;nbsp; pH 7.4+/- 0.1</td><td>Corning</td><td>21-040-CM</td><td></td></tr><tr><td>Pyridoxal 5-phosphate monohydrate, 99%&amp;nbsp;</td><td>Thermo Scientifc</td><td>228170010</td><td>1 g</td></tr><tr><td>UV-STAR MICROPLATE, 96 WELL, COC, F-BOTTOM</td><td>Greiner Bio</td><td>&amp;nbsp;655801</td><td></td></tr><tr><td>Wildtype Human Adenosine Deaminase 1</td><td>In-house</td><td></td><td></td></tr></tbody></table>

determining the serum stability of human adenosine deaminase 1 enzyme

The concept of enzyme stability is typically used to refer to an enzyme's thermostability - its ability to retain structure and activity as temperature increases. For a therapeutic enzyme, other measures of stability may also be critical, particularly its ability to retain function in human serum at 37 &#176;C, which we refer to as serum stability. Here, we describe an in vitro assay to assess the serum stability of the wildtype Homo sapiens adenosine deaminase I (HsADA1) enzyme using an absorbance-based microplate procedure. Specifically, this manuscript describes the preparation of buffers and reagents, a method arranging for the coincubation of HsADA1 in serum, a method to analyze the test samples using a microplate reader, and an accompanying analysis to determine the fraction of activity that an HsADA1 enzyme retains in serum as a function of time. We further discuss considerations to adapt this protocol to other enzymes, using an example of a Homo sapiens kynureninase enzyme, to help aid the protocol's adaptation to other enzymes where serum stability is of interest.

The figures show the results of the assay run when conducted with wildtype HsADA1. Figure 3A,B illustrate the absorbance decline curves at 260 nm of the samples originating from the 1x PBS/serum-enzyme mixtures for wildtype HsADA1 after the addition of adenosine. This declining absorbance as a function of time data is what the user may expect upon successful completion of the microplate-based assay and is similar to absorbance data that would arise after adding sufficient amounts of functional HsADA1 to adenosine. Figure 3C,D illustrates the absorbance curves of the tested negative controls, which contain no enzyme after adenosine addition. Compared to the actual enzyme samples, the absorbance change of these negative controls is almost negligible. From Figure 3, it can be seen that the slopes of the enzyme curves change as a function of incubation time. For example, the slope of the Day 0 timepoint is drastically steeper than the Day 5 timepoint.
Figure 4 illustrates the fraction of activity retained by wildtype HsADA1 as a function of time when incubated in either 1x PBS or pooled human serum at 37 &#176;C. Specifically, the slopes of all absorbance decline curves in Figure 3A,B are normalized to the Day 0 slopes from their respective groups. As can be seen in Figure 4, there is a divergence in the activity retained for wildtype HsADA1 in 1x PBS versus pooled human serum. Specifically, the enzyme loses its activity faster in pooled human serum, with the enzyme retaining approximately 10% of its original activity at Day 5. A loss of activity is still detectable when HsADA1 is incubated in 1x PBS, though the enzyme retains approximately 75% of its activity. This is congruent with Jennings et al.&#39;s results, also reporting a decrease in activity following incubation with serum at 37 &#176;C by almost 40% after 48 h6. Lu et al. have also shown that HsADA1 activity declines in pleural, peritoneal, and cerebrospinal bodily fluids at ambient temperature (25 &#176;C)8.
To demonstrate the flexibility of this protocol, we used it to test the serum stability of another enzyme of human origin, an engineered human kynureninase variant previously dubbed HsKYNase66-W102H-T333N7. HsKYNase66_W102H-T333N efficiently catalyzes the conversion of OH-Kyn into OH-AA, similar to the wildtype HsKYNase enzyme, although it is actually a two amino acid reversion mutant of an engineered human kynureninase variant with altered substrate specificity, i.e., that efficiently degrades kynurenine into anthranilic acid7. OH-Kyn absorbs strongly at 379 nm, while OH-AA does not, resulting in measurable absorbance decline upon the addition of our engineered HsKYNase variant to OH-kyn9,10. Figure 5A,B illustrates the absorbance decline curves at 379 nm of the samples originating from the 1x PBS/serum-enzyme mixtures for HsKYNase after the addition of OH-Kyn. Figure 5C,D illustrates the absorbance curves of the tested controls that do not contain enzymes. As seen with HsADA1, there is a clear difference in the slopes of the absorbance curves across samples. This is especially evident in Figure 5A, as the 0 h timepoint slope is much steeper than the 3 h and 24 h timepoints. Figure 6 illustrates the corresponding fraction of activity retained for the tested HsKYNase as a function of time. Like HsADA1, HsKYNase66-W102H-T333N loses activity in serum more quickly than in PBS (~85% activity lost versus ~40% over the course of 24 h). While this enzyme variant&#39;s serum stability has not been previously tested, it follows the general trend as other HsKYNase variants, which also lost most of their activity within 24 h in serum at 37 &#176;C7. 
Generally speaking, enzymes might lose activity over time due to a host of factors, including pH, salt concentration, temperature, and more11. Therefore, a result that does not follow this trend could be considered abnormal. For example, if the enzymatic activity increases drastically over time, this could indicate that the results are invalid. This is assuming enzyme conditions are not changed, and we are speaking strictly in a time sense.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67216/67216fig01.jpg" /> 
Figure 1: Flow chart detailing the steps of the protocol for HsADA1. The figure shows the steps followed in this protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67216/67216fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67216/67216fig02.jpg" /> 
Figure 2: Microplate reader kinetic read settings. A kinetic read protocol was created to measure the absorbance at 260 nm for 30 min at 37 &#176;C with the minimum interval. The figure depicts the chosen settings to set up this protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67216/67216fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67216/67216fig03.jpg" /> 
Figure 3: Absorbance versus time curves for HsADA1 after the addition of 200 &#181;M adenosine. (A)&#160;Absorbance versus time curves for serum-incubated HsADA1 after the addition of 200 &#181;M adenosine. (B)&#160;1x PBS-incubated HsADA1. (C) Serum negative control. (D) 1x PBS negative control (n =3). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67216/67216fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67216/67216fig04.jpg" /> 
Figure 4: HsADA1 activity retention. The activity is assessed in 1x PBS versus pooled human serum at 37 &#176;C. The fraction of original activity retained for HsADA1 incubated in 1x PBS versus pooled human serum over the course of 5 days was determined (bars = mean &#177; SD, n=3). A two-tailed unpaired t-test was used *p&#60;0.05, **p &#60; 0.01 and ***p &#60; 0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67216/67216fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67216/67216fig05.jpg" /> 
Figure 5: Absorbance versus time curves for HsKYNase66-W102H-T333N after the addition of 400 &#181;M OH-Kyn. (A)&#160;Absorbance versus time curves for serum-incubated HsKYNase after the addition of 400 &#181;M OH-Kyn. (B)&#160;1x PBS-incubated HsKYNase. (C) Serum negative control. (D) 1x PBS negative control (n=3). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67216/67216fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/67216/67216fig06.jpg" /> 
Figure 6: HsKYNase66-W102H-T333N activity retention. The activity is assessed in 1x PBS versus pooled human serum at 37&#160;&#176;C. The fraction of original activity retained for HsKYNase66-W102H-T333N incubated in 1x PBS versus pooled human serum over the course of 24 h was determined (bars = mean &#177; SD, n=3). A two-tailed unpaired t-test was used; ***p &#60; 0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67216/67216fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Standard curve of adenosine and inosine. The plot was drawn for 0-200 &#181;M. Adenosine or inosine was added to 1x PBS at the noted concentrations, and then the absorbance at 260 nm was measured. (n=3). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67216/Supplementary Figure 1.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Standard curve of OH-Kyn. The plot was drawn for 0-400 &#181;M.&#160;OH-Kyn was added to 1x PBS at the noted concentrations, and then the absorbance at 379 nm was measured. (n=3). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67216/Supplementary Figure 2.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3: Absorbance versus time curves at 260 nm with increasing concentrations of HsADA1 after the addition of 200 &#181;M adenosine. (A) Reaction curves for varying concentrations of HsADA1. (B) Fold change in the slope of the linear region of absorbance curves in relation to a 1x PBS control (bars = mean &#177; SD, n=3). A two-tailed unpaired t-test was used **p &#60; 0.01 and ****p &#60; 0.0001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67216/Supplementary Figure 3.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 4: Absorbance versus time curves at 379 nm with increasing concentrations of engineered HsKYNase after the addition of 400 &#181;M of OH-Kyn. (A) Reaction curves for varying concentrations of engineered HsKYNase. (B) Fold change in the slope of the linear region of absorbance curves in relation to a 1x PBS control (bars = mean &#177; SD, n=3). Two-tailed unpaired t-test was used ****p &#60; 0.0001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67216/Supplementary Figure 4.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 5: Absorbance versus time curves at 379 nm using heat-deactivated enzyme in 400 &#181;M of OH-Kyn. Heat-deactivated HsKYNase enzyme was added to 400 &#181;M of OH-Kyn in 1x PBS, and then the absorbance at 379 nm was measured. (n=3). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67216/Supplementary Figure 5.pdf" target="_blank">Please click here to download this File.</a>

Author Spotlight: Advancing Cellular and Protein Engineering to Control Biological Functions and Develop Novel Therapies

In this article, we detail methods to characterize an enzyme&#39;s ability to retain function when incubated at 37&#160;&#176;C in human serum, a pharmacological property referred to as its serum stability. This ability may be a key factor in predicting an enzyme&#39;s pharmacokinetic profile and its suitability for therapeutic use.

Determining the Serum Stability of Human Adenosine Deaminase 1 Enzyme

The concept of enzyme stability is typically used to refer to an enzyme's thermostability - its ability to retain structure and activity as temperature ...

determining-the-serum-stability-of-human-adenosine-deaminase-1-enzyme

Nanyang Technological University

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Research

JoVE Journal

Bioengineering

567 Views.  Georgia Institute of Technology. Ons lab werkt dus op het snijvlak van immunologie, engineering en metabolisme om de menselijke gezondheid te verbeteren. We gebruiken onze expertise op het gebied van eiwit- en cellulaire engineering om nieuwe therapieën te maken en biologische functies te controleren. Als onderzoekers de circulerende halfwaardetijd van biologische eiwitgeneesmiddelen willen verlengen, kan dit protocol gebruikers helpen informeren of serumstabiliteit een snelheidsbeperkende ...

Video: Auteur in de schijnwerpers: Bevordering van cellulaire en eiwittechnologie om biologische functies te beheersen en nieuwe therapieën te ontwikkelen

Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein substrates. The histone tails represent one of the most heavily modified stretches within all human proteins. Peptidyl-arginine deiminase 4 (PAD4) has been shown to convert arginine residues into the non-genetically encoded citrulline residue. Few assays described to date have been operationally facile with satisfactory sensitivity. Thus, the lack of adequate assays has likely contributed to the absence of potent non-covalent PAD4 inhibitors. Herein a novel fluorescence-based assay that allows for the monitoring of PAD4 activity is described. A pro-fluorescent substrate analog was designed to link PAD4 enzymatic activity to fluorescence liberation upon the addition of the protease trypsin. It was shown that the assay is compatible with high-throughput screening conditions and has a strong signal-to-noise ratio. Furthermore, the assay can also be performed with crude cell lysates containing over-expressed PAD4.

PAD4 is an enzyme responsible for the conversion of peptidyl-arginine to peptidyl-citrulline. Dysregulation of PAD4 has been implicated in a number of human diseases. A facile and high-throughput compatible fluorescence based PAD4 assay is described.

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein ...

Chemistry

Investigating Protein Sequence-structure-dynamics Relationships with Bio3D-web

We demonstrate the usage of Bio3D-web for the interactive analysis of biomolecular structure data. The Bio3D-web application provides online functionality for: (1) The identification of related protein structure sets to user specified thresholds of similarity; (2) Their multiple alignment and structure superposition; (3) Sequence and structure conservation analysis; (4) Inter-conformer relationship mapping with principal component analysis, and (5) comparison of predicted internal dynamics via ensemble normal mode analysis. This integrated functionality provides a complete online workflow for investigating sequence-structure-dynamic relationships within protein families and superfamilies.

A protocol for the online investigation of protein sequence-structure-dynamics relationships using Bio3D-web is presented.

We demonstrate the usage of Bio3D-web for the interactive analysis of biomolecular structure data. The Bio3D-web application provides online functionality ...

Biochemistry

Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further supported by recent findings in high-resolution mass spectrometry based proteomics showing that many new proteins and sites within these proteins can be acetylated. However the identity of the enzymes regulating these proteins and sites is often unknown. Among these enzymes, sirtuins, which belong to the class III histone lysine deacetylases, have attracted great interest as enzymes regulating the acetylome under different physiological or pathophysiological conditions. Here we describe methods to link SIRT2, the cytoplasmic sirtuin, with its substrates including both in vitro and in vivo deacetylation assays. These assays can be applied in studies focused on other members of the sirtuin family to unravel the specific role of sirtuins and are necessary in order to establish the regulatory interplay of specific deacetylases with their substrates as a first step to better understand the role of protein acetylation. Furthermore, such assays can be used to distinguish functional acetylation sites on a protein from what may be non-regulatory acetylated lysines, as well as to examine the interplay between a deacetylase and its substrate in a physiological context.

Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates

This protocol describes the required steps to execute in vitro and in vivo deacetylation assays in order to establish the role of proteins as specific deacetylation substrates for sirtuins and further study the role of reversible - lysine acetylation as a post-translational modification.

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further ...

HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and validated for the quantification of extracellular purine nucleotides and nucleosides produced by purified chronic lymphocytic leukemia (CLL) cells under different culture conditions. The chromatographic separation of adenosine 5&#39;-monophosphate (AMP), adenosine (ADO) and inosine (INO) is performed at RT on a silica-based, reversed-phase column that is used for polar compound retention. The method includes a binary mobile phase, which consists of 7 mM ammonium acetate and acetonitrile with a flow rate of 1.00 ml/min. The eluates are monitored using a Photodiode Array UV detector set at 260 nm. A standard calibration curve is generated to calculate the equation for the analytical quantification of each purine compound. System control, data acquisition and analysis are then performed. Applying this protocol, AMP, INO and ADO elute at 7, 11 and 11.9 min, respectively, and the total run time for each sample is 20 min. This protocol may be applied to different cell types and cell lines (both suspension and adherent), using culture media as matrix. The advantages are easy and fast sample preparation and the requirement of a small amount of supernatant for analysis. Furthermore, the use of a serum-free medium allows skipping the protein precipitation step with acetonitrile that impacts the final concentration of purine compounds. One of the limitations of the method is the requirement of the equilibration column run before each single sample run, making the total run time of the experiment longer and preventing high throughput screening applications.

The protocol described here represents an easy and reproducible method that employs reverse phase high-performance liquid chromatography (RP-HPLC) to measure purine metabolism on chronic lymphocytic leukemia (CLL) cells cultured under different conditions.

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and ...

Measuring Enzymatic Stability by Isothermal Titration Calorimetry

This work demonstrates a new method for measuring the stability of enzyme activity by isothermal titration calorimetry (ITC). The peak heat rate observed after a single injection of the substrate solution into an enzyme solution is correlated with enzyme activity. Multiple injections of the substrate into the same enzyme solution over time show the loss of enzyme activity. The assay is autonomous, requiring very little personnel time, and is applicable to most media and enzymes.&#160; &#160;

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.

This work demonstrates a new method for measuring the stability of enzyme activity by isothermal titration calorimetry (ITC). The peak heat rate observed ...

Environment

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Enabling Real-Time Compensation in Fast Photochemical Oxidations of Proteins for the Determination of Protein Topography Changes

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based structural biology technique that probes the solvent-accessible surface area of proteins. This technique relies on the reaction of amino acid side chains with hydroxyl radicals freely diffusing in solution. FPOP generates these radicals in situ by laser photolysis of hydrogen peroxide, creating a burst of hydroxyl radicals that is depleted on the order of a microsecond. When these hydroxyl radicals react with a solvent-accessible amino acid side chain, the reaction products exhibit a mass shift that can be measured and quantified by mass spectrometry. Since the rate of reaction of an amino acid depends in part on the average solvent accessible surface of that amino acid, measured changes in the amount of oxidation of a given region of a protein can be directly correlated to changes in the solvent accessibility of that region between different conformations (e.g., ligand-bound versus ligand-free, monomer vs. aggregate, etc.) FPOP has been applied in a number of problems in biology, including protein-protein interactions, protein conformational changes, and protein-ligand binding. As the available concentration of hydroxyl radicals varies based on many experimental conditions in the FPOP experiment, it is important to monitor the effective radical dose to which the protein analyte is exposed. This monitoring is efficiently achieved by incorporating an inline dosimeter to measure the signal from the FPOP reaction, with laser fluence adjusted in real-time to achieve the desired amount of oxidation. With this compensation, changes in protein topography reflecting conformational changes, ligand-binding surfaces, and/or protein-protein interaction interfaces can be determined in heterogeneous samples using relatively low sample amounts.

Fast photochemical oxidation of proteins is an emerging technique for the structural characterization of proteins. Different solvent additives and ligands have varied hydroxyl radical scavenging properties. To compare the protein structure in different conditions, real-time compensation of hydroxyl radicals generated in the reaction is required to normalize reaction conditions.

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based structural biology technique that probes the solvent-accessible surface area ...

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

Neuroscience

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya6. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila7-14. An acetate kinase which can only utilize PPi but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus15,16.
In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD+ by the enzymes pyruvate kinase and lactate dehydrogenase21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine23. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP+ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase24.
Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PPi.

A method for the determination of acetate kinase activity is described. This assay utilizes a direct reaction for determining enzyme activity and kinetics of acetate kinase in the acetate-forming direction with different phosphoryl acceptors. Furthermore, this method can be utilized for assaying other acetyl phosphate or acetyl-CoA utilizing enzymes.

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

Biology

In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

The use of personalized medicine to treat rare monogenic diseases like lysosomal storage disorders (LSDs) is challenged by complex clinical trial designs, high costs, and low patient numbers. Hundreds of mutant alleles are implicated in most of the LSDs. The diseases are typically classified into 2 to 3 different clinical types according to severity. Moreover, molecular characterization of the genotype can help predict clinical outcomes and inform patient care. Therefore, we developed a simple cell culture assay based on HEK293H cells heterologously over-expressing the mutations identified in Fabry and Pompe disease. A similar assay has recently been introduced as a preclinical test to identify amenable mutations for Pharmacological Chaperone Therapy (PCT) in Fabry disease. This manuscript describes an amended cell culture assay which enables rapid phenotypic assessment of allelic variants in Fabry and Pompe disease to identify eligible patients for PCT and may aid in the development of novel pharmacochaperones.

In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

There is a demand to make pre-clinical testing for a novel class of "orphan" drugs called pharmacological chaperones reproducible, fast, and efficient. We developed a simple, highly standardized, and versatile cell culture-based assay to screen for eligible patients as well as novel pharmacological chaperone drugs.

The use of personalized medicine to treat rare monogenic diseases like lysosomal storage disorders (LSDs) is challenged by complex clinical trial designs, ...

Determining the Serum Stability of Human Adenosine Deaminase 1 Enzyme

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