An Amplex Red-based fluorometric and spectrophotometric assay for l-asparaginase using its natural substrate

Abstract

Leukemia L-Aspartate oxidase Coupled enzyme assay Amplex Red a b s t r a c tWe report on the development of a sensitive real-time assay for monitoring the activity of L-asparaginase that hydrolyzes L-asparagine to L-aspartate and ammonia. In this method, L-aspartate is oxidized by Laspartate oxidase to iminoaspartate and hydrogen peroxide (H 2 O 2 ), and in the detection step horseradish peroxidase uses H 2 O 2 to convert the colorless, nonfluorescent reagent Amplex Red to the red-colored and highly fluorescent product resorufin. The assay was validated in both the absorbance and the fluorescence modes. We show that, due to its high sensitivity and substrate selectivity, this assay can be used to measure enzymatic activity in human serum containing L-asparaginase.Ó 2013 Elsevier Inc. All rights reserved.The enzyme L-asparaginase (EC 3.5.1.1, L-asparagine amidohydrolase, L-ASNase), 1 which predominantly occurs in microorganisms and plants, catalyzes the hydrolysis of L-asparagine (L-Asn) to L-aspartic acid (L-Asp) and ammonia [1,2]. Escherichia coli L-ASNase has been used extensively as a therapeutic enzyme in the frontline treatment of lymphoblastic malignancies, such as acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma [3,4], since the 1960s. In light of the significance of L-ASNase as a therapeutic protein, various methods have been developed for measuring the enzyme's activity. Those assays can be used in either absorbance mode [5][6][7][8][9][10] or fluorescence mode [11,12]. However, these assays suffer from certain disadvantages that are primarily related to the use of substrate analogs instead of the natural substrate L-Asn [6,7,11,12]. Such assays are not suitable for in vitro evolution of L-asparaginases that aims to select variants showing improved catalytic efficiency and selectivity for the physiological substrate. Moreover, the limited sensitivity of absorption-based spectrophotometric assays [5][6][7]9,10] is a major handicap to significantly reducing reaction volumes. A noteworthy example of such a case is droplet-based microfluidics, which has emerged as a powerful tool for high-throughput screening in directed protein evolution [13,14]. In these experimental setups, the assay volume is minimized to 1 nl, to 1 fl, scaling down light path lengths to 1 lm.Here, we report on the development of a novel L-ASNase assay that can be used in either the fluorescence or absorbance mode and relies solely on the use of the physiological substrate L-Asn. In this three-step coupled enzyme system (Fig. 1), L-Asp, which is one of the two products of the L-ASNase reaction, is oxidized by L-aspartate oxidase (L-AspOx), resulting in the formation of iminoaspartate and hydrogen peroxide (H 2 O 2 ); the latter product is used by horseradish peroxidase (HRP) to oxidize the nonfluorescent compound Amplex Red (AR) to resorufin, which exhibits excellent fluorescence as well as absorbance properties.For establishing and quantitatively evaluating the assay, we cloned and recombinantly produced E. coli L-AspOx [15]...

Development of L-Asparaginase Biobetters: Current Research Status and Review of the Desirable Quality Profiles

“…However, this method has certain limitations such low sensitivity and the fact that it do not measure intracellular ASNase activity (Vaishali and Bhupendra, 2017). After the initial screening, other methods are required for the quantitative evaluation of enzymatic activity such as the aforementioned Nessler's reaction (Peterson and Ciegler, 1969); the L-aspartyl-β-hydroxamic acid (AHA) method, wich evaluates aspartyl β-hydroxamate formation after L-asparagine hydrolysis in the presence of hydroxylamine (Frohweinm et al, 1971); circular dichroism spectroscopy (Kudryashova and Sukhoverkov, 2016); amplex Red method (Karamitros et al, 2014), among others. The screening methods for finding enzymes with reduced glutaminase activity are similar to the plate methods used for ASNase activity detection, i.e., they are based on pH switch, but use GLN instead of ASN as a substrate.…”

From Compartmentalization of Bacteria within Inorganic Macrocellular Beads to the Assembly of Microbial Consortia

“…We measure and compare the kinetics for bead stoichiometries ranging from 1:0 to 1:3. The cascade reaction becomes faster upon increasing the number of aspartate oxidase beads (Figure f), in qualitative agreement with the observation in bulk that the reaction is limited by the amount of aspartate oxidase . We note that the background increase observed in the case of beads containing only the asparaginase‐overexpressing cells is likely to arise from the presence of aspartate oxidase endogenously expressed by our E. coli strains.…”

Ginkgo biloba extract protects human neuroblastoma SH‑SY5Y cells against oxidative glutamate toxicity by activating redoxosome‑p66Shc

“…ROS concentration was analyzed using the fluorescence microscope (Leica Microsystems). Intracellular H 2 O 2 level was measured by Amplex Red assay as previously reported (17,18).…”