**2. SODs**

#### **2.1. The structure and function of SOD**

Superoxide dismutases (SODs, EC 1.15.1.1) are ubiquitous and one important class of antiox‐ idant metalloenzymes against the harmful effects of superoxide free radicals. The main function of SODs was to decompose superoxide radicals into molecular oxygen and hydrogen peroxide inside cells, which reaction is as follows:

$$2\text{O}\_2^{\cdot -} + 2\text{H}^+ \rightarrow \text{O}\_2 + \text{H}\_2\text{O}\_2 \tag{1}$$

Based on the functional metal cofactors located at the active sites, four distinct classes of SODs have been found (**Figure 1**). SODs of Class I specifically require manganese or iron ion for catalytic activity (MnSOD and FeSOD) and enzymes that function with either of the two metal ions so‐called cambialistic SODs. Class II is copper‐ and zinc‐dependent enzymes (CuZn‐ SODs). Members of the two classes are found in both prokaryotes and eukaryotes. Nickel‐ containing SODs are mainly identified from marine actinomycetes and cyanobacteria [5, 6]. Enzymatic reactions of SODs depending on different metals indicate that SODs are developed by cells to offset the harmful effects of reactive oxygen species and match its surroundings.

Due to their antioxidative effects, SODs exhibited dramatic potential in medicine, cosmetic, food, agriculture, and chemistry industry. For example, considerable clinical experiments have shown that SODs could prevent oncogenesis and reduce the cytotoxic effects of anticancer drugs. Recently, SODs have found to prevent many diverse diseases such as cardiovascular diseases, diabetes, asthma, infertility, neurological disorders, and transplant rejection. SODs have also been successfully utilized as a major component in cosmetics for skin protection. In animal husbandry, SODs are considered to be one kind of strong antioxidative enzymes, which can reduce the oxidative stress of animal and prevent the oxidation of animal products, improve the quality of animal products such as meat, egg, or milk. In order to make better use of SODs, their characteristics, the most suitable environment and influencing factors, etc., should be known especially the information of inhibitor and activator.

**Figure 1.** A comparison of the enzyme structures and active sites for the four SODs, (a) streptomyces coelicolor NiSOD (PDB, 1t6u), (b) human Cu/ZnSOD (PDB, 1pu0), (c) *E. coli* FeSOD (PDB, 1isa), and (d) MnSOD (PDB, 1vew). (adapted from reference [7]).

#### **2.2. Inhibitors of SODs**

turnover rates,is the main enzyme involved in reduction of H2O2 via the Fenton reaction. This enzyme is almost exclusively expressed in peroxisomes [2]. GPX is specific for its hydrogen donor, but nonspecific for H2O2, and it degrades H2O2 using reduced glutathione in a powerful

From the perspective of protection, improving antioxidase activity would be helpful for organisms to survive under various stresses, but from another respect, the antioxidase activity should be inhibited. Some anticancer agents, such as xenobiotics and radiation, act by pro‐ ducing ROS to kill tumor cells. Cells with high levels of antioxidant enzymes are resistant to these anticancer agents. Therefore, the levels of cellular antioxidant enzymes will influence the sensitivity of tumor cells to anticancer therapies [4]. Thus, more detailed information about an activator and inhibitor of SOD, CAT, and GPX should be known, for better utilization of these

Superoxide dismutases (SODs, EC 1.15.1.1) are ubiquitous and one important class of antiox‐ idant metalloenzymes against the harmful effects of superoxide free radicals. The main function of SODs was to decompose superoxide radicals into molecular oxygen and hydrogen

Based on the functional metal cofactors located at the active sites, four distinct classes of SODs have been found (**Figure 1**). SODs of Class I specifically require manganese or iron ion for catalytic activity (MnSOD and FeSOD) and enzymes that function with either of the two metal ions so‐called cambialistic SODs. Class II is copper‐ and zinc‐dependent enzymes (CuZn‐ SODs). Members of the two classes are found in both prokaryotes and eukaryotes. Nickel‐ containing SODs are mainly identified from marine actinomycetes and cyanobacteria [5, 6]. Enzymatic reactions of SODs depending on different metals indicate that SODs are developed by cells to offset the harmful effects of reactive oxygen species and match its surroundings. Due to their antioxidative effects, SODs exhibited dramatic potential in medicine, cosmetic, food, agriculture, and chemistry industry. For example, considerable clinical experiments have shown that SODs could prevent oncogenesis and reduce the cytotoxic effects of anticancer drugs. Recently, SODs have found to prevent many diverse diseases such as cardiovascular diseases, diabetes, asthma, infertility, neurological disorders, and transplant rejection. SODs have also been successfully utilized as a major component in cosmetics for skin protection. In animal husbandry, SODs are considered to be one kind of strong antioxidative enzymes, which can reduce the oxidative stress of animal and prevent the oxidation of animal products, improve the quality of animal products such as meat, egg, or milk. In order to make better use

O H O HO <sup>2</sup> 2 22 2 2 -× + + ®+ (1)

manner [3].

208 Enzyme Inhibitors and Activators

enzymes.

**2. SODs**

**2.1. The structure and function of SOD**

peroxide inside cells, which reaction is as follows:

#### *2.2.1. Different inhibitors on sensitivity of SODs*

Cu/Zn‐SODs, Fe‐SODs, and Mn‐SODs are sensitive to different inhibitors, and we could distinguish these metal‐specific SODs based on different inhibitory reactions (**Table 1**). The activity of Cu/Zn‐SODs was inhibited by KCN, but Fe‐SODs and Mn‐SODs were not sensitive to KCN. Whereas, completely different phenomenon was observed when incubated in chloroform‐ethanol (1:3, V/V) component solvent, as a result Fe‐SODs and Mn‐SODs activity were almost lost, while Cu/Zn‐SOD was not sensitive to chloroform‐ethanol. Fe‐SODs and Mn‐ SODs are highly homologous and exhibit structural similarity, it is assumed that they originate from the same ancestry [8]. Fe‐SODs and Mn‐SODs can be distinguished by their different sensitivities to H2O2, because Mn‐SOD activity was not sensitive to H2O2. In addition, NaN3 also is used to detect the type of MnSOD, when the SOD was inhibited by neither KCN nor H2O2 [9].


"+" indicate reaction and "‐" indicate no reaction.

**Table 1.** Effect of inhibitors on different cofactors of SODs activity.

#### *2.2.2. Effects of metal ions on SOD activity*

Cu2+, Zn2+, Fe2+, and Mn2+ are cofactors of superoxide dismutase, and they are vital to enzymatic activity. But heterogeneous expressed SODs were often existed in terms of apoenzyme or combined other metals present in a culture medium [10], thus might result in partial or total loss of activity. So that reconstituted metal ion is necessary to recovery activity. Apoenzyme was prepared by a metal removal procedure according reference [11] and simplified as follow:


To be attention, only specific metal ions combined to SOD, the activity could be recovered. But for Fe‐SOD and Mn‐SOD, because they share highly homologous, Fe‐substituted Mn‐SODs also are active, but the activity is lower than Mn‐reconstituted SOD.

SODs are metalloenzyme, and they will be inhibited by chelators [12, 13], such as EDTA and cuprizone (a copper‐specific chelator). They should be avoided losing metal cofactors caused by chelators in the experiment. But a Mn‐SOD from *Mycobacterium* sp. JC1 DSM 3803 was not sensitive to 10 mM EDTA [8], which may be due to the highly tightness of metal cofactors binding to enzymes.

Co2+, Hg2+, K+ and, Al3+ and other metal ions also show their inhibition effects on SOD activity. A Mn‐SOD from deep‐sea thermophile *Geobacillus* sp. EPT3 was activated only by Mn2+ among nine tested metal ions [12]. A Cu‐/Zn‐SOD from black soybean was also activated only by Cu2+ [13] and a similar result was shown in the report of Liu et al. [14]. But there are a few exceptions, for example, a manganese‐containing superoxide dismutase was activated by Cu2+, Zn2+, and Al3+.

### *2.2.3. Singlet molecular oxygen inactivation of superoxide dismutase*

High reactive singlet molecular oxygen (1 O2) is one kind of short‐lived intermediate from oxidation reaction which oxidizes a variety of biological molecules easily, including lipids, nucleic acids, and proteins, and it also promotes deleterious processes such as lipid peroxida‐ tion, membrane damage, and cell death [15]. The biochemical production of singlet oxygen has been proposed to contribute to the destructive effects on a number of biological processes [16]. *In vivo*, singlet molecular oxygen is produced under normal and pathophysiological conditions. It is known to be particularly reaction with histidine, which often located at the active sites of SODs, so singlet molecular oxygen may result in prevention of the activities of those enzymes including CATs and GPXs. *In vitro*, singlet molecular oxygen could be produced by photoactived dyes, such as methylene blue or rose bengal [17], so inactivation of SODs, CATs, and GPXs should be avoid in application.

#### **2.3. Different activator on activity of SODs**

*2.2.2. Effects of metal ions on SOD activity*

for 18 h.

210 Enzyme Inhibitors and Activators

binding to enzymes.

Cu2+, Zn2+, and Al3+.

Co2+, Hg2+, K+

Cu2+, Zn2+, Fe2+, and Mn2+ are cofactors of superoxide dismutase, and they are vital to enzymatic activity. But heterogeneous expressed SODs were often existed in terms of apoenzyme or combined other metals present in a culture medium [10], thus might result in partial or total loss of activity. So that reconstituted metal ion is necessary to recovery activity. Apoenzyme was prepared by a metal removal procedure according reference [11] and simplified as follow: **1.** Purified SOD was dialyzed against denature buffer: 20 mM 8‐hydroxyquinoline, 2.5 mM

**2.** Purified SOD was dialyzed against 5 mM Tris buffer (pH 7.8), containing 1 mM metal ions

To be attention, only specific metal ions combined to SOD, the activity could be recovered. But for Fe‐SOD and Mn‐SOD, because they share highly homologous, Fe‐substituted Mn‐SODs

SODs are metalloenzyme, and they will be inhibited by chelators [12, 13], such as EDTA and cuprizone (a copper‐specific chelator). They should be avoided losing metal cofactors caused by chelators in the experiment. But a Mn‐SOD from *Mycobacterium* sp. JC1 DSM 3803 was not sensitive to 10 mM EDTA [8], which may be due to the highly tightness of metal cofactors

A Mn‐SOD from deep‐sea thermophile *Geobacillus* sp. EPT3 was activated only by Mn2+ among nine tested metal ions [12]. A Cu‐/Zn‐SOD from black soybean was also activated only by Cu2+ [13] and a similar result was shown in the report of Liu et al. [14]. But there are a few exceptions, for example, a manganese‐containing superoxide dismutase was activated by

oxidation reaction which oxidizes a variety of biological molecules easily, including lipids, nucleic acids, and proteins, and it also promotes deleterious processes such as lipid peroxida‐ tion, membrane damage, and cell death [15]. The biochemical production of singlet oxygen has been proposed to contribute to the destructive effects on a number of biological processes [16]. *In vivo*, singlet molecular oxygen is produced under normal and pathophysiological conditions. It is known to be particularly reaction with histidine, which often located at the active sites of SODs, so singlet molecular oxygen may result in prevention of the activities of those enzymes including CATs and GPXs. *In vitro*, singlet molecular oxygen could be produced by photoactived dyes, such as methylene blue or rose bengal [17], so inactivation of SODs,

and, Al3+ and other metal ions also show their inhibition effects on SOD activity.

O2) is one kind of short‐lived intermediate from

guanidinium chloride, 5 mM Tris, 0.1 mM EDTA, pH 3.8, at 4°C for 18 h.

**3.** Excess metal ions were removed by dialysis against 5 mM Tris buffer (pH 7.8).

also are active, but the activity is lower than Mn‐reconstituted SOD.

*2.2.3. Singlet molecular oxygen inactivation of superoxide dismutase*

High reactive singlet molecular oxygen (1

CATs, and GPXs should be avoid in application.

#### *2.3.1. Effect of carbohydrates on activities of superoxide dismutase*

Carbohydrates, such as maltose, sucrose, lactose, trehalose, glucose, d‐fructose, d‐trehalose, d‐xylose, and so on could stabilize an enzyme structure. For example, trehalose plays a strong promotive effect on superoxide dismutase [18]. Trehalose is a kind of polyol compound with many hydroxyl groups, which has strong hydration ability and can change the free energy to the favorable direction in solution. The multihydroxyl structure of trehalose can connected with both the surface of the enzyme protein and the external water through hydrogen bonding, so that the structure of the enzyme is stable, and the enzyme activity was protected [19].

#### *2.3.2. Polyethylene glycol modified SOD to improve its efficiency*

Polyethylene glycols (PEGs) are considered to be a safety and nonimmunogenic materials. They also have multihydroxyl compounds and can be activated by many activators, such as cyanuric, dicycolhexylcarbodiimide, N‐hydroxysucciniimide, and 1,1'‐carbonyldiimidazole, then activated polyethylene glycol was conjugated with the ε‐NH2 group of SOD. PEG conjugated with SOD not only enhance the stability of the enzyme, but also avoid enzymatic immunogenicity. For example, Beckman et al. [20] reported that a superoxide dismutase conjugated with polyethylene glycol greatly increased endothelial the cell oxidant resistance and half‐life of the enzyme.

#### *2.3.3. Cyclodextrin modified SOD to enhance its stability*

Cyclodextrins (CD), cyclic oligosaccharides containing six (α‐CD), seven (β‐CD) or eight (γ‐CD) α‐1‐4‐linked d‐glucopyranose units have been used to stabilize enzymes in order to increase their activities and favor immobilization. On the one hand, superoxide dismu‐ tase modified by β‐cyclodextrin could improve its performance. A superoxide dismutase was glycosylated by cyclodextrin‐branched carboxymethylcellulose and its plasma half‐life time was prolonged from 4.8 min to 7.2 h, its anti‐inflammatory activity also increased by 2.2 times [21]. On the other hand, cyclodextrin and its derivative could synthesize SOD mimics. Puglisi et al. [22] reported a 6A,6B‐Dideoxy‐6A,6B‐di[(N‐salicylidene)amino]‐β‐cy‐ clodextrin conjugated with a manganese(III) complex showed a SOD‐like activity and a good solubility that favor its application.
