Determining the Serum Stability of Human Adenosine Deaminase 1 Enzyme

Summary

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.

Abstract

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 °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.

Introduction

The following method allows a user to quantitatively assess an enzyme'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 °C and time-coursed analyses of retention of enzyme activity. We refer to an enzyme'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's substrate and the resulting product. The concept of se....

Protocol

1. Serum incubation

1. Prepare a 10x stock of HsADA1 in 1x PBS pH 7.4 (1x PBS) at a final concentration of 10 µM. Thaw a 15 mL aliquot of pooled human serum and pre-warm it to 37 °C. Prewarm a 50 mL aliquot of 1x PBS to 37 °C.
2. Prepare the enzyme-serum incubation mixtures by adding 100 µL of the 10x HsADA1 stock to 900 µL of pooled human serum in a low-bind microcentrifuge tube, referred to as the enzyme + serum mixture. In a separate low-bind microcentrifuge tu.......

Discussion

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 nm^6,8,12,13. This assay is inspired by several previous works. Kalackar, for ex.......

Materials

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