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Purification and characterization of a psychrophilic glutathione reductase from Antarctic ice microalgae Chlamydomonas sp. Strain ICE-L

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Abstract

A psychrophilic glutathione reductase from Antarctic ice microalgae Chlamydomonas sp. Strain ICE-L was purified by ammonium sulfate fractionation and three steps of chromatography. The yield was up to 25.1% of total glutathione reductase in the crude enzyme extract. The glutathione reductase activity was characterized by the spectrophotometric method under different conditions. Purified glutathione reductase was separated by SDS-PAGE, which furnished a homogeneous band. The native molecular mass of the enzyme was 115 kDa. Apparent Km values for NADPH and NADH (both at 0.5 mmol L−1 oxidized glutathione) were 22.3 and 83.8 μmol L−1, respectively. It was optimally active at pH 7.5, and it was stable from pH 5 to 9. Its optimum temperature was 25°C, with activity at 0°C 23.5% of the maximum. Its optimum ion strength and optimum Mg2+ were 50–90 and 7.5 mmol L−1, respectively. Ca2+, Mg2+, and cysteine substantially increased the activity of the enzyme but chelating agents, heavy metals (Cd2+, Pb2+, Cu2+, Zn2+, etc.), NADPH, and ADP had significant inhibitory effects. This glutathione reductase can be used to study the adaptation and mechanism of catalysis of psychrophilic enzymes, and it has a high potential as an environmental biochemical indicator under extreme conditions.

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Abbreviations

DTT:

Dithiothreitol

EGTA:

1,2-Di-(2-aminoethoxy)ethane-N,N,N′,N′-tetraaetic acid

GR:

Glutathione reductase

GSH:

Reduced glutathione

GSSG:

Oxidized glutathione

NEM:

N-ethylmaleimide

PVP:

Polyvinylpyrrolidone

SDS:

Sodium dodecyl sulfate

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Acknowledgments

This work was supported by the Natural Science Foundation of China (NSFC, No. 40206022; No. 40406003).

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Authors and Affiliations

  1. Fisheries College, Guangdong Ocean University, No. 40 Jiefang East Road, Zhanjiang, Guangdong, 524025, People’s Republic of China

    Yu Ding, Ji-Chang Jian & Zao-He Wu

  2. Key Laboratory of Marine Bio-active Substances, First Institute of Oceanography, State Oceanic Administration, Qingdao, Shandong, 266061, People’s Republic of China

    Jin-Lai Miao, Quan-Fu Wang, Zhou Zheng & Guang-You Li

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  1. Yu Ding
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  2. Jin-Lai Miao
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Correspondence to Yu Ding.

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Ding, Y., Miao, JL., Wang, QF. et al. Purification and characterization of a psychrophilic glutathione reductase from Antarctic ice microalgae Chlamydomonas sp. Strain ICE-L. Polar Biol 31, 23–30 (2007). https://doi.org/10.1007/s00300-007-0328-5

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