Detection of hydrogen peroxide in Photosystem II (PSII) using catalytic amperometric biosensor.

Ankush Prasad,Mika Tada,Masaki Kobayashi,Aditya Kumar,Makoto Suzuki,Tomoya Sugai,Hiroyuki Kikuchi,Shigenobu Kasai,Pavel Pospíšil

doi:10.3389/fpls.2015.00862

Abstract

Hydrogen peroxide (H2O2) is known to be generated in Photosystem II (PSII) via enzymatic and non-enzymatic pathways. Detection of H2O2 by different spectroscopic techniques has been explored, however its sensitive detection has always been a challenge in photosynthetic research. During the recent past, fluorescence probes such as Amplex Red (AR) has been used but is known to either lack specificity or limitation with respect to the minimum detection limit of H2O2. We have employed an electrochemical biosensor for real time monitoring of H2O2 generation at the level of sub-cellular organelles. The electrochemical biosensor comprises of counter electrode and working electrodes. The counter electrode is a platinum plate, while the working electrode is a mediator based catalytic amperometric biosensor device developed by the coating of a carbon electrode with osmium-horseradish peroxidase which acts as H2O2 detection sensor. In the current study, generation and kinetic behavior of H2O2 in PSII membranes have been studied under light illumination. Electrochemical detection of H2O2 using the catalytic amperometric biosensor device is claimed to serve as a promising technique for detection of H2O2 in photosynthetic cells and subcellular structures including PSII or thylakoid membranes. It can also provide a precise information on qualitative determination of H2O2 and thus can be widely used in photosynthetic research.

Highlights

Photosystem II (PSII) is a multi-subunit pigment-protein complex which is located in the thylakoid membrane of chloroplasts of cyanobacteria, algae and higher plants that comprises of more than 25 proteins and the concomitant cofactors (Ferreira et al, 2004; Loll et al, 2005; Guskov et al, 2009; Kawakami et al, 2011)
We used spectroscopic and amperometric techniques to measure the production of reactive oxygen species (ROS) in spinach PSII membrane
Our prime objective was to study the effect of high light stress in PSII membrane reflected by ROS production primarily the generation of H2O2 as the stress response formed via the dismutation of O2−

Summary

Introduction

Photosystem II (PSII) is a multi-subunit pigment-protein complex which is located in the thylakoid membrane of chloroplasts of cyanobacteria, algae and higher plants that comprises of more than 25 proteins and the concomitant cofactors (Ferreira et al, 2004; Loll et al, 2005; Guskov et al, 2009; Kawakami et al, 2011). Chlorophyll pigments of the PSII antenna complex absorb light energy and use it for the oxidation of water molecules and reduction of plastoquinone. Light energy absorbed by chlorophyll pigments converted into the energy of separated charges and consequent water-plastoquinone oxidoreductase activity is involuntarily linked with the production of ROS (Pospíšil, 2009, 2012). In unison, released molecular oxygen serves as a forerunner of ROS, which at low concentration play an important role in cell regulation, whereas if formed in excess, is responsible for oxidation of biomolecules such as lipid, proteins, and nucleic acid (Halliwell and Gutteridge, 2007). Direct oxidation of proteins and lipids by UV irradiation and toxic chemicals following subsequent chemical reactions are known to be associated with formation of ROS (Halliwell and Gutteridge, 2007; Prasad and Pospíšil, 2012)

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Conclusion