Voltammetric appraisal of PON1’s activities: a preliminary study

Abstract

Introduction: The human serum paraoxonase 1 (EC. 3.1.8.1, PON1) is a high-density lipoprotein associated endogenous free-radical scavenging system with arylesterase (ARase), lactonase (LOase) and paraoxonase (POase) activities [1]. PON1 has been linked to several pathological conditions, such as atherosclerosis, cardiovascular diseases, and neurodegenerative disorders [1]. Additionally, it acts as a protective agent against xenobiotic toxicity, through its ability to hydrolyse organophosphate derivates [1]. Therefore, the monitorization of each PON1 activity is of great interest in clinical, environmental and warfare contexts. Electrochemical-based sensing methods are ideal for PON1 activity assessment since many of the involved reactants are electroactive. Furthermore, these methods are highly attractive for point-of-care testing applications and on-site analysis, due to their simplicity, low-cost, sensitivity, selectivity and a high degree of miniaturization. Previously, we reported a cyclic voltammetry assay for POase activity determination using ethyl-paraoxon as substrate [2]. We now turn to the LOase and AREase activities, using homocysteine thiolactone (HcyTl) and phenylacetate (PA) as the respective substrates [3,4], for the appraisal of PON1 status by voltammetric techniques.

Materials and methods: Recombinant wild-type like PON1 (rePON1) G3C9 was expressed in E. coli BL21 and purified according to [5]. In the electrochemical assays, pyrolytic graphite, glassy carbon and screen-printed carbon electrodes were used as working electrodes in a three-electrode configuration with Ag/AgCl reference and platinum/carbon counter electrodes. Enzymatic reactions were analyzed between the potential window (−0.8; 1 V), at 50 mV s⁻¹.

Results: The hydrolysis of HcyTl in the presence of rePON1 (LOase activity) was not observed in any condition tested. In contrast, PA was found to be quickly hydrolysed, as showed by an increase in the oxidation current intensity associated to the product formation (phenol).

Discussion and conclusions: These preliminary results show that in wild-type like rePON1 G3C9, the AREase activity is the one with the highest turn-over, since the hydrolysis of the PA occurred almost instantly, while the reaction with ethyl-paraoxon (POase) took a few minutes [2] to produce a measurable current signal variation, and the hydrolysis of HcyTl (LOase) was not measurable at all, over a time course of 18 hours.

Acknowledgements

This work was supported by the Applied Molecular Biosciences Unite-UCIBIO which is financed by national funds from FCT/MCTES (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728). from UCIBIO@REQUIMTE Pest-C/EQB/LA0006/2013. The authors acknowledge the financial support from Fundação para a Ciência e Tecnologia (PD/BD/109687/2015).