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Purification of Glutathione S-Transferase pi from Erythrocytes and Evaluation of the Inhibitory Effect of Hypericin

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Abstract

Hypericin is a photosensitizer compound used in the photodynamic therapy (PDT). PDT is an alternative cancer treatment strategy whose function is dependent on the photosensitizers accumulating selectively in tumor cells and following visible or infra-red light induced activation lead to the apoptosis/necrosis of the tumor cells via the formation of reactive oxygen species. Thus, the cellular redox balance is essential for the efficacy of PDT. Among the protective enzyme systems glutathione S-transferases (GST, E.C.2.5.1.18) function in detoxification, protection against oxidative stress and intracellular transport of molecules. It is known that isoenzymes of GST and especially GST-pi is increased in cancer cells and it plays very important functions in the development of resistance to anticancer drugs. Since photosensitizers are used intravenously, it is important to elucidate the effects of photosensitizers on the erythrocyte enzymes. The aim of the present study was to investigate the impact of hypericin on human erythrocyte GST-pi (heGST-pi). Purification yield of 71 % and purification fold of 2550 were achieved by using conventional chromatographic methods. The specific activity of the enzyme is found as 51 U/mg protein. Hypericin inhibited heGST-pi in a dose dependent manner and inhibition was biphasic. Noncompetitive type of inhibition was observed with both substrates, GSH and CDNB. The inhibitory constant (K i ) values obtained from Lineweaver–Burk, Dixon, secondary plots; slope and y-intercept versus 1/S (substrate) and from non-linear regression analysis were in good correlation: K i (GSH) was calculated as 0.19 ± 0.01 μM and K i (CDNB) as 0.26 ± 0.03 μM.

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Abbreviations

CDNB:

1-Chloro-2,4-dinitrobenzene

CYP:

Cytochrome P450

EA:

Ethacrynic acid

ECL:

Enhanced chemilluminescence

EDTA:

Ethylenediaminetetraacetic acid

GSH:

Reduced glutathione

GST:

Glutathione S-transferase

heGST-pi:

Human erythrocyte GST-pi

K i :

Inhibitory constant

K m :

Michaelis constant

PB74:

Polybuffer 74

PBE-94:

Polybuffer exchanger 94

PDT:

Photodynamic therapy

PVDF:

Polyvinyl difluoride

RBC:

Red blood cells

ROS:

Reactive oxygen species

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

V m :

Maximum velocity

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Acknowledgments

We gratefully thank to Dr. Yasemin Aksoy and Dr. Yaman Muşdal for their support to carry out Western blotting experiments. We also thank to Dr. Umut Gazi for language editing of the manuscript. This work was supported by Hacettepe University Scientific Research Council (HUBAB-011D04101002).

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Authors and Affiliations

  1. Department of Medical Biochemistry, Faculty of Medicine, Hacettepe University, 06100, Ankara, Turkey

    Seyhan Turk & Gulnihal Kulaksiz Erkmen

  2. Department of Medical Biochemistry, Faculty of Medicine, Near East University, Nicosia, 99138, Mersin 10, Turkey

    Ozlem Dalmizrak, I. Hamdi Ogus & Nazmi Ozer

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  1. Seyhan Turk
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  2. Gulnihal Kulaksiz Erkmen
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  3. Ozlem Dalmizrak
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Correspondence to Nazmi Ozer.

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Turk, S., Kulaksiz Erkmen, G., Dalmizrak, O. et al. Purification of Glutathione S-Transferase pi from Erythrocytes and Evaluation of the Inhibitory Effect of Hypericin. Protein J 34, 434–443 (2015). https://doi.org/10.1007/s10930-015-9638-6

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