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 tu...

Evaluating Serum Stability of HsADA1: A Microplate-Based Approach

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalytically+active+holo+homo+sapiens+adenosine+deaminase+i+adopts+a+closed+conformation.">Catalytically active holo homo sapiens adenosine deaminase i adopts a closed conformation.</a> Acta Crystallograph Sect D. 78 (1), 91-103 (2022).</li><li>Li, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+4+nucleosides+via+one+reference+compound+in+chinese+cordyceps+by+hplc-uv+at+equal+absorption+wavelength.">Determination of 4 nucleosides via one reference compound in chinese cordyceps by hplc-uv at equal absorption wavelength.</a> Natural Prod Comm. 18 (3), 1934578X231161410 (2023).</li><li>Whitmore, K. V., Gaspar, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenosine+deaminase+deficiency+-+more+than+just+an+immunodeficiency.">Adenosine deaminase deficiency - more than just an immunodeficiency.</a> Front Immunol. 7, 314 (2016).</li><li>Jennings, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+expression+and+purification+of+a+human+adenosine+deaminase+in+E.+coli+and+characterization+of+its+asp8asn+variant.">Optimized expression and purification of a human adenosine deaminase in E. coli and characterization of its asp8asn variant.</a> Prot Express Purificat. 213, 106362 (2024).</li><li>Blazeck, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bypassing+evolutionary+dead+ends+and+switching+the+rate-limiting+step+of+a+human+immunotherapeutic+enzyme.">Bypassing evolutionary dead ends and switching the rate-limiting step of a human immunotherapeutic enzyme.</a> Nat Catalysis. 5 (10), 952-967 (2022).</li><li>Lu, J., Grenache, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+rapid,+microplate-based+kinetic+assay+for+measuring+adenosine+deaminase+activity+in+body+fluids.">Development of a rapid, microplate-based kinetic assay for measuring adenosine deaminase activity in body fluids.</a> Clinica Chimica Acta. 413 (19), 1637-1640 (2012).</li><li>Maciel, L. G., Dos Anjos, J. V., Soares, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+low-cost+evaluation+of+hydroxykynurenine+activity.">Fast and low-cost evaluation of hydroxykynurenine activity.</a> MethodsX. 7, 100982 (2020).</li><li>Bokman, A. H., Schweigert, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-hydroxyanthranilic+acid+metabolism.+Iv.+Spectrophotometric+evidence+for+the+formation+of+an+intermediate.">3-hydroxyanthranilic acid metabolism. Iv. Spectrophotometric evidence for the formation of an intermediate.</a> Arch Biochem Biophys. 33 (2), 270-276 (1951).</li><li>Tanford, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in protein chemistry. 23, 121-282 (1968).</li><li>Tritsch, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validity+of+the+continuous+spectrophotometric+assay+of+kalckar+for+adenosine+deaminase+activity.">Validity of the continuous spectrophotometric assay of kalckar for adenosine deaminase activity.</a> Anal Biochem. 129 (1), 207-209 (1983).</li><li>Gracia, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+catalytic+site+structural+gate+of+adenosine+deaminase+allosterically+modulates+ligand+binding+to+adenosine+receptors.">The catalytic site structural gate of adenosine deaminase allosterically modulates ligand binding to adenosine receptors.</a> FASEB J. 27 (3), 1048-1061 (2013).</li><li>Kalckar, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+spectrophotometry+of+purine+compounds+by+means+of+specific+enzymes:+Iii.+Studies+of+the+enzymes+of+purine+metabolism.">Differential spectrophotometry of purine compounds by means of specific enzymes: Iii. Studies of the enzymes of purine metabolism.</a> J Bio Chem. 167 (2), 461-475 (1947).</li><li>Hartwick, R., Jeffries, A., Krstulovic, A., Brown, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+assay+for+adenosine+deaminase+using+reverse+phase+high+pressure+liquid+chromatography.">An optimized assay for adenosine deaminase using reverse phase high pressure liquid chromatography.</a> J Chromatographic Sci. 16 (9), 427-435 (1978).</li><li>Paul, M. K., Grover, V., Mukhopadhyay, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Merits+of+hplc-based+method+over+spectrophotometric+method+for+assessing+the+kinetics+and+inhibition+of+mammalian+adenosine+deaminase.">Merits of hplc-based method over spectrophotometric method for assessing the kinetics and inhibition of mammalian adenosine deaminase.</a> J Chromatography B. 822 (1), 146-153 (2005).</li><li>Ubbink, J. B., Vermaak, W. J. H., Bissbort, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-performance+liquid+chromatographic+assay+of+human+lymphocyte+kynureninase+activity+levels.">High-performance liquid chromatographic assay of human lymphocyte kynureninase activity levels.</a> J Chromatography B: Biomed Sci Appl. 566 (2), 369-375 (1991).</li><li>Tsentalovich, Y. P., Snytnikova, O. A., Forbes, M. D. E., Chernyak, E. I., Morozov, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemical+and+thermal+reactivity+of+kynurenine.">Photochemical and thermal reactivity of kynurenine.</a> Exp Eye Res. 83 (6), 1439-1445 (2006).</li><li>Demain, A. L., Vaishnav, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+recombinant+proteins+by+microbes+and+higher+organisms.">Production of recombinant proteins by microbes and higher organisms.</a> Biotechnol Adv. 27 (3), 297-306 (2009).</li><li>Gr&#228;slund, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+production+and+purification.">Protein production and purification.</a> Nat Methods. 5 (2), 135-146 (2008).</li><li>Faber, M. S., Whitehead, T. 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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 ex...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adenosine&#160;</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&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>13-698-795</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific&#160;</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&#160;</td>
			<td>TCI</td>
			<td>I0037</td>
			<td>25 g</td>
		</tr>
		<tr>
			<td>PBS, 1x&#160; pH 7.4+/- 0.1</td>
			<td>Corning</td>
			<td>21-040-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridoxal 5-phosphate monohydrate, 99%&#160;</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>&#160;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.

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

Determining the Serum Stability of Human Adenosine Deaminase 1 Enzyme

So our lab works at the interface of immunology, engineering, and metabolism to improve human health. We use our expertise in protein and cellular engineering to make new therapies and control biological functions. If researchers desire to increase the circulating half-life of protein biologics, this protocol could help inform users whether serum stability is a rate limiting step.Understanding this key point would help guide future protein engineering efforts to the area of need. One question we'll explore is whether proteins with enhanced serum stability result in greater therapeutic efficacy in the context of treating cancer than their non engineered counterparts. We'll further explore whether therapeutic efficacy can be achieved using less doses due to this enhanced stability.

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 am...

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.

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

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Preparation of Serum Samples for Stability Evaluation of Human Adenosine Deaminase 1 Enzyme Using Microplate Assay

To begin, prepare a 10x stock of HsADA1 in 1x PBS at pH 7.4 to achieve a final concentration of 10 micromolar. Prewarm a thawed 15-milliliter aliquot of pooled human serum to 37 degrees Celsius. Then add 100 microliters of the 10x HsADA1 stock to both 900 microliters of pooled human serum and PBS taken in two separate low-bind microcentrifuge tubes.Dilute 100 microliters of each enzyme and non-enzyme mixture one to one with 30%glycerol made in 1x PBS. Flash-freeze the tubes with liquid nitrogen and store at minus 80 degrees Celsius. Seal all the tubes with transparent film and place them in a 37 degrees Celsius incubator.

Absorbance-Based Microplate Analysis to Test the Serum Stability of Human Adenosine Deaminase 1

To begin, prepare the serum and enzyme mixtures along with their respective controls. In the microplate reader, set up a kinetic read protocol to measure the absorbance at 260 nanometers with the shortest reading interval for 30 minutes. Next, thaw all the frozen samples on ice and dilute each thawed sample 1 to 10 with 1x PBS pre-warmed to 37 degrees Celsius in LoBind microcentrifuge tubes.To prepare a five millimolar adenosine stock, add 66.8 milligrams of adenosine to 50 milliliters of 1x PBS. After diluting it to a 250 micromolar solution, pre-warm the adenosine to 37 degrees Celsius in an incubator. To a 96-well ultraviolet compatible microplate, add 160 microliters of the adenosine followed by 40 microliters of each diluted protein sample in triplicates.Measure the absorbance of each well at 260 nanometers for 30 minutes at 37 degrees Celsius. For data analysis, export the data to a spreadsheet, then determine the slope of the linear region of the absorbance data as a function of time for the first several minutes. Subtract the slope of the negative control consisting of pooled human serum or PBS from the slopes of the enzyme plus serum mixtures and the enzyme plus PBS mixtures respectively.Finally, normalize all the adjusted slopes to the original slope at the zero hour time point to obtain the fraction of activity remaining using the equation, and plot the fraction of activity remaining versus time. Wildtype HsADA1 activity decreased more rapidly in pooled human serum compared to 1x PBS over five days. The absorbance decline curves for wildtype HsADA1 showed a steeper initial slope at day zero compared to day five.Negative control samples showed negligible absorbance change compared to enzyme samples.

298 Views.  Georgia Institute of Technology.  In this article, we detail methods to characterize an enzyme's ability to retain function when incubated at 37 °C in human serum, a pharmacological property referred to as its serum stability. This ability may be a key factor in predicting an enzyme's pharmacokinetic profile and its suitability for therapeutic use.

Evaluating Serum Stability of HsADA1: A Microplate-Based Approach

Summary