Activity determination of human monoamine oxidase B (Mao B) by selective capturing and amperometric hydrogen peroxide detection

Highlights

• An electrochemical detection system for a Parkinson’s disease target enzyme - Mao B - has been developed.

• Selective capturing of Mao B from a sample solution is achieved via specific antibodies.

• The antibody binding is not disturbing the enzyme activity of Mao B.

• Enzymatically produced H₂O₂ is quantified with Prussian blue carbon electrodes.

• The system provides sufficient sensitivity to meet biological activities.

Abstract

The enzyme monoamine oxidase B (Mao B), which is involved in dopamine metabolism, is an important drug target in Parkinson’s disease (PD). The medical treatment with several Mao B inhibitors is well established but only little personalized since the monitoring of the patients Mao B activity is complex and requires sophisticated laboratory equipment. To demonstrate an approach, which has potential in the personalization of the medical PD treatment, a sensorial Mao B activity determination system has been developed. Here, the enzyme activity is quantified by electrochemical detection of enzymatically produced H₂O₂. Therefore, the enzyme is enriched from solution via cellulose particles with specific antibodies against human Mao B. The successful capturing of the enzyme can be verified by means of SDS-PAGE. For activity determination the enzyme is brought in contact with a suitable substrate - here benzylamine. Selectivity of the amperometric hydrogen peroxide detection in the presence of co-reactants has been verified. Within the time span of 30 min, a linear dependency of enzymatically produced H₂O₂ with the substrate incubation time can be observed. This allows the evaluation of the Mao B activity. The results have been correlated to an optical detection method. Furthermore, the method has been tested for different amounts of enzyme used in the experiments.

Introduction

Parkinson’s disease (PD) is one of the most common neurodegenerative disorders worldwide. It affects about 0.3 % of the global population and approximately 2 % of people older than 80 years [1]. PD is mainly characterized by the degeneration of neurons in certain areas of the substantia nigra [2]. The loss of neurons leads to a dopamine deficiency in the brain, which induces disturbances in the signal transmission at synapses and therefore movement disorders occur.

Currently, there is no therapy available that can stop the progression of PD or prevent its manifestation. However, to improve the patient’s quality of life, several symptomatic therapies including inter alia pharmacotherapy to adjust the dopamine concentration have been established [3]. In the early stage of PD the pharmacotherapy with a dopamine precursor (L-Dopa) and inhibitors for dopamine degrading enzymes leads to a constant clinical response and significant relief of the symptoms [3]. After the initial period of the therapy with continuous clinical response (1–3 years), motor fluctuations appear and intensify with the progression of the disease, attributed to the further degeneration of dopaminergic cells [3,4]. To improve the medical treatment of PD, the dosage of medication could be adjusted or at least the duration of action of a dose could be extended. Therefore, biosensors monitoring the enzyme activity of target enzymes or other clinical parameters have the potential to contribute to further develop the symptomatic treatment of PD.

In this study we have focused on the enzyme activity determination of Mao B, which catalyzes the oxidative deamination of primary and secondary aromatic amines such as dopamine [5]. However, it should be added here that also other enzymes (catechol-O-methyl transferase and dopa-decarboxylase) are involved in dopamine metabolism and are therefore target enzymes for PD drugs. With respect to this, the electrochemical enzyme activity determination for catechol-O-methyl transferase has been demonstrated recently by selective dopamine detection [6].

In literature several methods for Mao B activity determination have been described. For the enzyme activity measurement of tissue homogenates or platelets from blood samples radiometric assays were very frequently used with specific ¹⁴C labeled Mao B substrates [[7], [8], [9], [10], [11], [12], [13]], since this method allows a specific and sensitive quantification in complex media. However, radiometric devices are unsuitable for the development of easy-to-use systems for medical purpose since they require special permits and safety precautions. Furthermore, fluorometric assays detecting enzymatically produced H₂O₂ with a peroxidase reaction and a fluorescent dye were reported several times for Mao B activity determination [[14], [15], [16]]. However, fluorometric assays for Mao B activity analysis are disadvantageously for clinical applications since they suffer from poor selectivity in complex biological fluids.

In contrast to this, relatively few publications have focused on electrochemical quantification of Mao B activity. Reyes-Parada et al. have demonstrated Mao B activity determination with HPLC and electrochemical detection. In this approach a crude rat brain mitochondrial suspension (containing Mao B) was incubated with 4-dimethylaminophenethylamine as a specific substrate and 4-dimethylaminophenylacetic acid was detected as the product of the enzymatic reaction by HPLC with amperometric detection [17]. Here oxidation of 4-dimethylaminophenylacetic acid was used. A linear dependency of product formation with the substrate incubation time was observed (Reyes-Parada et al., 1994). With this method a good sensitivity has been achieved, but chromatographic separation and equipment is necessary. Thus, we focus in our investigations on a more sensorial orientated approach.

Following the example of the blood glucose meter, the enzyme activity determination of Mao B is intended to be performed by specific electrodes. This concept is based on the selective detection of one component of the reaction mixture in the presence of all the others. For Mao B reaction there are several possibilities in principle. One direction can be seen in electrodes, which can discriminate the educt from the product of the reaction. Here, some progress has been achieved by a proper choice of the electrode material avoiding additional layers on the sensor [18]. Furthermore the selective detection of a Mao B substrate (dopamine) in the presence of its metabolic product (3,4-dihydroxyphenylacetic acid) could be demonstrated exploiting a novel voltammetric technique, multiple cyclic square wave voltammetry with PEDOT:NAFION coated carbon-fiber microelectrodes [19]. Although there is considerable work on sensorial catecholamine detection in the literature [[20], [21], [22], [23], [24], [25]] - even in the presence of interfering substances [[26], [27], [28]], the selective detection of catecholamines in the presence of their metabolites has not been devoted much attention in research. Another direction can, however, be seen by exploiting H₂O₂ which is generated by the enzyme during substrate conversion.

Here, Prussian blue electrodes have been used to detect H₂O₂. Prussian blue is an important material for the H₂O₂ detection owing to its pseudo peroxidase activity, which allows to reduce H₂O₂ at low overpotentials (−50 mV vs. Ag/AgCl) in the presence of O₂ [29,30]. Prussian blue is a ferric ferrocyanide (Fe₄^III [Fe^II(CN)₆)]₃) with the iron(III) atom coordinated to nitrogen and the iron(II) coordinated to carbon [29,31]. When Prussian blue is reduced Prussian white is formed, which can be oxidized reversibly to Prussian blue [29,[32], [33], [34], [35]]. The mechanism of H₂O₂ reduction involves the transfer of two electrons from Prussian white to H₂O₂ forming two hydroxide ions [36]. Prussian blue owns a good catalytic specificity for H₂O_2, because of its polycrystalline structure, which facilitates only the penetration of small molecules as H₂O₂ into the lattice, while larger molecules such as ascorbic acid, uric acid or para-acetylaminophenol are excluded [37]. These advantageous sensing properties are intended to be combined here with a specific antibody-based capturing to quantify the Mao B activity.