Efficiency of superoxide anions in the inactivation of selected dehydrogenases

https://doi.org/10.1016/j.radphyschem.2010.04.001Get rights and content

Abstract

The most ubiquitous of the primary reactive oxygen species, formed in all aerobes, is the superoxide free radical. It is believed that the superoxide anion radical shows low reactivity and in oxidative stress it is regarded mainly as an initiator of more reactive species such as radical dotOH and ONOO.

In this paper, the effectiveness of inactivation of selected enzymes by radiation-generated superoxide radicals in comparison with the effectiveness of the other products of water radiolysis is examined. We investigate three enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH).

We show that the direct contribution of the superoxide anion radical to GAPDH and ADH inactivation is significant. The effectiveness of the superoxide anion in the inactivation of GAPDH and ADG was only 2.4 and 2.8 times smaller, respectively, in comparison with hydroxyl radical. LDH was practically not inactivated by the superoxide anion.

Despite the fact that the studied dehydrogenases belong to the same class of enzymes (oxidoreductases), all have a similar molecular weight and are tetramers, their susceptibility to free-radical damage varies. The differences in the radiosensitivity of the enzymes are not determined by the basic structural parameters analyzed. A significant role in inactivation susceptibility is played by the type of amino acid residues and their localization within enzyme molecules.

Introduction

The superoxide anion radical demonstrated considerably smaller reactivity with proteins in comparison to the radical dotOH radical, and therefore in some papers its reaction with proteins is neglected (Bielski and Cabelli, 1995, Bielski et al., 1985, Schüssler and Puchala, 2004, Gebicki, 2006). The lower reactivity of O2radical dot is connected with its longer half-life time in comparison to the hydroxyl radical. In effect, it can diffuse at great distances and react with amino acid residues, which are the most important for protein functioning. If these residues are situated inside or near the protein’s active site, the superoxide radical can also cause significant inactivation of the enzyme.

Moore et al. (2000) in the work on the radiolysis of protein A estimated that superoxide is as efficient as the hydroxyl radical in this protein inactivation. Similar observations regarding alcohol dehydrogenase were made by Badiello et al. (1974).

The aim of this paper was to estimate the effectiveness of the superoxide radical in the inactivation of selected enzymes in comparison to the effectiveness of other products of water radiolysis. It was also attempted to find a connection between the structure of the examined enzymes and their sensitivity to free-radical inactivation.

Section snippets

Materials

Alcohol dehydrogenase (ADH) (alcohol:NAD+ oxidoreductase, EC 1.1.1.1), molecular weight of 147 kDa from baker’s yeast, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (d-glyceraldehyde-3-phosphate: NAD+ oxidoreductase, EC 1.2.1.12), molecular weight of 143 kDa from a rabbit muscle, lactate dehydrogenase (LDH) (l-lactate:NAD+ oxidoreductase, EC 1.1.1.27), molecular weight of 140 kDa from a rabbit muscle, xanthine oxidase (XO) (xanthine:oxygen oxidoreductase, EC 1.17.3.2) from bovine milk, xanthine

Efficiency of radiation-generated radicals in inactivation of dehydrogenases

In this paper, the radiation inactivation of three enzymes: GAPDH, ADH and LDH was determined. The solutions of enzymes were irradiated under air with doses of up to 120 Gy in four systems:

  • 1

    in the absence of other compounds;

  • 2

    in the presence of mannitol;

  • 3

    in the presence of mannitol and SOD and

  • 4

    in the presence of mannitol, SOD and catalase.

The dependence of log (% activity) on the radiation dose for the studied systems is presented in Fig. 1, Fig. 2, Fig. 3. In all the cases, these dependences are

Discussion

The superoxide anion radical is the most ubiquitous reactive form of oxygen in all aerobic organisms. Its main sources are: mitochondria, neutrophils, hemoglobin autooxidation processes and reactions of numerous enzymes including xanthine oxidase. It is believed that superoxide anion radical shows low reactivity and in the oxidative stress it is regarded mainly as an initiator of more reactive species such as radical dotOH and ONOO (Gebicki, 2006). Several papers devoted to the direct influence of O2radical dot on

References (28)

  • P. Bartholmes et al.

    Molecular properties of lactic dehydrogenase under the conditions of the enzymatic test. Sedimentation analysis and gel filtration in the microgram and nanogram range

    Eur. J. Biochem.

    (1973)
  • B.H.J. Bielski et al.

    Superoxide and hydroxyl radical chemistry in aqueous solutions

  • B.H.J. Bielski et al.

    Reactivity of HO2/O2 radicals in aqueous solution

    J. Phys. Chem. Ref. Data

    (1985)
  • R.K. Bonnichsen et al.

    Liver alcohol dehydrogenase

  • Cited by (0)

    1

    Fax: +48 426354473.

    View full text