Büro für Altlastenerkundung und Umweltforschung

Dr. Rainer Haas

Stadtwaldstr. 45a, D-35037 Marburg, Tel.: 06421/93084, Fax: 06421/93073

email: haasr@gmx.net

Degradation of Phenarsarzinchloride by manganese peroxidase

Rainer Haas1, Olga Tsivunchyk2,4, Klaus Steinbach3, Eberhard v. Löw4, Katrin Scheibner5, Martin Hofrichter6

1: Office of environmental investigation and research, Stadtwaldstrasse 45a, D-35037 Marburg, Germany

2: Yanka Kupala Grodno State University, Ozheshko str. 22, Grodno, 230023, Republic of Belarus

3: Philipps University Marburg, Dept. of Chemistry, Hans-Meerwein-Str., D-35037 Marburg, Germany

4: Institute of Immunology, Philipps University Marburg, Pilgrimstein 2, D- 35037 Marburg, Germany

5: JenaBios GmbH, Löbstedter Strasse 78, D-07749 Jena, Germany

6: Lehrstuhl für Umweltbiotechnologie, Internationales Hochschulinstitut Zittau, Markt 23, D-02763 Zittau, Germany

Corresponding author: Dr. Rainer Haas


The investigations with manganese peroxidase for the destruction of arsenoorganic chemical warfare agent adamsite (phenarsarzine chloride) were done. The results show that adamsite is metabolized within 48 h to 100%. During the degradation of adamsite another peak occures in the HPLC chromatogram. The UV spectra of the both peaks are similar. With HPLC/MS no identification was possible. Because of short retention time and UV spectra we guess, that a metabolite with As(V) is built.

Key words: adamsite; analyses; analyses, HPLC; arsenic; chemical warfare agents; chemical warfare agents, destruction; cwa; enzymatic combustion; HPLC; manganese peroxidase; mass spectrometry; phenarsarzine chloride

1 Introduction

Manganese peroxidase is well known as enzyme that is able to destroy lignin, it is used by mushrooms for the decomposition of lignin. Former investigations showed the possibility to destroy a lot of persistent organical compounds e.g nitro aromatic compounds or halogen containing compounds [1,2]. Some arsenoorganical compounds were used as chemical warfare agents. Especially aromatic arsenoorganic compounds are very stable and persistent in the environment. Experiments showed that it was possible to destroy this persistent molecules with a cell free manganese peroxidase (MnP) system in laboratory scale [3]. Analyses for these experiments were done with gas chromatography [4].

The chemical warfare agent phenarsarzine chloride (adamsite) cannot be analyzed with gas chromatographical method. The destruction of adamsite with MnP was detected with HPLC and diode array detector.

2 Experimental

The MnP degredation experiments were done in 20 ml vials. The degradation rate was determined in comparison with a zero solution without manganese peroxidase. The double experiments were done.

The following chemicals were added consistently:

- 3 ml Na-Malonate buffer (c = 100 mmol/l, pH 4.5)

- 0.6 ml MnCl2 solution (c = 20 mmol/l)

- 0.3 ml glutathione (GSH) solution (c = 100 mmol/l)

- 1.0 ml Methanol p.a.

- 0.3 ml MnP solution (activity: 60 U/ml) or instead of MnP solution 0.3 ml water for zero experiment


- 500 µl Adamsit solution (c = 1 mg/ml).

The reaction vials are stored at room temperature. After 2 h, 4 h, 8 h, 24 h, 48h, 72 h and 144 h 0.5 ml of this solution is given to 0.5 ml methanol in 1.4 ml vials for HPLC chromatography. After preparation the vials were frozen.

The following cross experiment was done: in the second experiment after 1h reaction time a vial was filled and diluted with 50% Methanol. This vial was measured 2 h after filling with HPLC and then additionally after 1 day, 1.5 days and 2 days. This experiment was done to find out, if reaction with MnP was stopped after filling in vials and diluting with 50% methanol. The cross experiment was necessary, because every HPLC analysis needed 75 min and vials were stored in autosampler before analysis for 20 h maximum.

The HPLC analyses were done with a HPLC Gynkotek system, Germering (Germany) with the following components:

pump: M 480; degaser: GT-103; autosampler: GINA 50; column thermostate at 20 °C with column oven STH 585; diode array detector: UVD 340-S, spectral resolution: 2 nm; data acquisition and system controlling: software Gynkosoft V 5.50.

Column: Nucleosil 120 RP 18, 5 µm, 250 * 3 mm with precolumn 10 * 3 mm; injection volume: 50 µl; eluent: methanol/water; flow: 0,5 ml/min; flow program: pre-running (15 min): methanol/water 20/80, from 0 min to 30 min gradient methanol/water 20/80 to 80/20; then in 1 min to 100% methanol; 100% methanol (29 min).

Wavelength of detection: simultaneous 210 nm and 230 nm; UV spektrum from 190 nm - 400 nm.

HPLC/MS analyses for the identification of adamsite metabolite were done under the same HPLC conditions. Mass spectrometric coupling was done with the electrospray method.

3 Results and discussion

The peak area of adamsite and adamsite metabolite in the vial after 1 h reaction time was stable within a data acquisition time of 48 h. The result of the cross experiment shows, that reaction of manganese peroxidase degradation is stopped after dilution of the reaction solution with 50% methanol.

The experiment with zero solution shows that there is no degradation of adamsite after 144 h. In zero experiment no adamsite metabolite is detected.

Additional investigations with coupled HPLC and mass spectrometry were done to identify this suspect adamsite reaction product. These investigations were not successful.

The degradation experiments with MnP show, that adamsite is metabolized by MnP to 100% after 48h. The percentage amount of adamsite was calculated in relation to zero experiment (czero = 100%). The percentage amount of adamsite metabolite was calculated with the commitance that after 48 h adamsite was totally metabolized. Under this consumptions the calculated sum of adamsite and metabolite was found from 89% to 101% of the original concentration. The reuslts are shown in table 1.

During degradation of adamsite (retention time: 35.7 min) with HPLC method another peak occures (retention time: 8.5 min). The UV-spectra of both peaks are very similar. The figure 1 shows the UV spectra of adamsite and the metabolite. It was assumpted that this compound was a polar reaction product of adamsite, because of less retention time. We do consider, that polar functions (e.g. hydroxy groups) are not added at the aromatic ring, so as the result would be a remacable changing of the UV spectra. According to the fact that the UV spectra are similar we guess that arsenic in adamsite molecule is oxidized from As(III) to As(V).

Table 1: Results of adamsite degradation by MnP after HPLC analysis, rest concentration of adamsite and metabolite in % and concentration of adamsite in reaction solution in µg/ml; Zero experiment: Adamsite = 90 µg/ml


adamsite in %

metabolite in %

adamsite (µg/ml)

metabolite (µg/ml)









































nd: not detectable

4 References

[1] M. Hofrichter, K. Vares, K. Scheibner, G. Galkin, J. Sipliae, A. Hatakka: Mineralization and solubilization of synthetic lignin (DHP) by manganese peroxidases from Nematoloma frowardii and Phlebia radiata. Journal of Biotechnology 67 (1999), 217 - 228

[2] M. Hofrichter, K. Scheibner, I. Schneegass, W. Fritsche: Enzymatic combustion of Aromatic and Aliphatic Compounds by Manganese Peroxidase from Nematoloma frowardii. Applied and Environmental Microbiology 64 (2) (1998), 399 - 404

[3] R. Haas, K. Scheibner, M. Hofrichter: Enzymatische Umsetzung von Arsenkampfstoffen durch das Pilzenzym Mangan-Peroxidase. UWSF - Z. Umweltchem. Oekotox. in print

[4] R. Haas, A. Krippendorf, T.C. Schmidt, K. Steinbach, E.V. Löw: Chemisch-analytische Untersuchung von Arsenkampfstoffen und ihren Metaboliten. UWSF - Z. Umweltchem. Ökotox. 10 (1998), 289 - 293

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