Bovine erythrocyte SOD

Probably more is known about bovine erythrocyte SOD than about any copper protein (Table I), and it is the only one for which x-ray structural information is available. This chapter reviews the salient structural and mechanistic features of the enzyme, focusing on the active site. There are additional reviews of the literature in several excellent articles (6,7,8,17). 

Table 6.3 displays the solution pKa values of the promoters capable of facilitating the electron transfer of Cu, Zn-SOD listed in Table 6.1. The —COOH-termi-nated SAMs are mostly negatively charged in phosphate buffer (pH 7.0). Although the bovine erythrocyte Cu, Zn-SOD has a net negative charge at pH 7.0 (p/ = 4.9), an electrostatic interaction is still expected to occur between the SAMs and the positively charged amino acid moieties (typically —NI13). Besides, the hydrogen bonding between —COOH groups and the amino acid residues is believed to comprehensively...

The formal potentials ( ° ) of the three kinds of SODs were found to be dependent on solution pH as displayed in Fig. 6.6. As shown, the formal potential of bovine erythrocyte Cu, Zn-SOD decreases linearly with increasing solution pH with a slope of ca. -60mV/pH from pH 5.8 to pH 9.5 (curve b), indicating one proton and one electron are included in the electrode reaction of Cu, Zn-SOD, which is similar to previously proposed enzymatic catalytic mechanistic scheme of the Cu, Zn-SOD [139— 144], In contrast, the pH dependency of Fe-SOD from E. coli was complicated (curve a) the formal potential changes linearly with solution pH in a range from pH 5.8 to 8.5 with a slope of ca. -60mV/pH, and becomes pH-independent at above pH > 8.5. Previous studies have observed that the Fe (III) form of the protein ionizes with an apparent pKa of 9.0 0.3 and such ionization effect has been interpreted in terms of hydrolysis of a bound water molecule with p/<"a of ca. 8.5 [145], The C -pII profile of... 

SOD, isolated from bovine erythrocytes, is a blue-green protein due to the presence of copper, removal of which by treatment with EDTA results in loss of activity, which is restored by adding Cu2+ it also contains Zn2+, which does not appear to be at the active site. The enzyme, which is very stable in 9 M urea at neutral pH, consists of two identical subunits of molecular weight 16kDa held together by one or more disulphide bonds. The amino acid sequence has been established. 

Milk contains trace amounts of SOD which has been isolated and characterized it appears to be identical to the bovine erythrocyte enzyme. SOD inhibits lipid oxidation in model systems. The level of SOD in milk parallels that of XO (but at a lower level), suggesting that SOD may be excreted in milk in an attempt to offset the pro-oxidant effect of XO. However, the level of SOD in milk is probably insufficient to explain observed differences in the oxidative stability of milk. The possibility of using exogenous SOD to retard or inhibit lipid oxidation in dairy products has been considered. 

HP here is great interest in the biochemistry and relevant coordination chemistry of copper-containing proteins (1,2, 3, 4, 5). They are widely distributed in both plants and animals and are often involved in oxygen metabolism, transport, and use. One of the most actively studied copper proteins is bovine erythrocyte superoxide dismutase (SOD) (6,7,8). This enzyme catalyzes the dismutation of superoxide ion, Reaction 1. 

Superoxide Dismutase Origin bovine erythrocytes Roche Diagnostics Superoxide Dismutase (SOD)... 

The Mn(ii)-based SOD mimics have numerous potential advantages over the SOD enzymes as potential therapeutic agents, including membrane permeability, selective reactivity for superoxide, immunogenicity effects, stability, and cost. Table 1 compares characteristics of the Mn(ii)-based SOD mimics with R = H versus those of bovine erythrocyte Cu,Zn SOD. 

Characteristic Mn- / HN, l -Nh Bovine erythrocyte Cu, Zn SOD enzyme Advantage of SOD mimic... 

Bovine erythrocyte Cu,Zn SOD protected against the superoxide-mediated injury to the endothelial cells, but the results were quite variable, and a bellshaped dose-response curve was observed. However, the SOD mimics SC-52608 and SC-54417 showed reproducible dose-dependent protection against the injury, with nearly complete inhibition being obtained with 150/zM complex. SC-54385, a Mn(ii) complex that has no detectable SOD activity, did not protect the cells against the superoxide-mediated injury, even at concentrations as high as 300 /zM. These results are consistent with human neutrophil-mediated injury to aortic endothelial cells being mediated by superoxide. 

Figure 3 Ribbon of the Cu,Zn-SOD of bovine erythrocytes, based on the coordinates of Tainer et al. (2SOD.pdb) All molecular structures are depicted using Molscript. ...

Figure 4 Active site of Cu,Zn-SOD, based on the oxidized-state coordinates of Tainer et al. (2SOD.pdb, bovine erythrocytes) and the reduced state coordinates of Ogihara et al (IJCV.pdb, from yeast). The Cu ions are shown in green and Zn in black, other atoms are colored according to cpk. The reduced state structure is depicted using heavy bonds and darkened colors including C atoms in gray, the oxidized state structure is depicted with light bonds and light-colored atoms, including white C atoms.

An intriguing phenomenon was observed, when the purified bovine erythrocyte Cu ZnjSuperoxide dismutases, obtained by the different isolation techniques were compared. Thermal stability measurements revealed that the purified SOD s are much less heat resistant compared to the enzymes in the homogenates. Therefore, heat deterioration of the isolated protein must not be connected with the isolation technique described in Chapter 2.3. When freshly aqueously isolated Cu ZnjSuper-oxide dismutase was heated to 77 C a transient and marked increase of the specific enzymic activity is seen. 

Fig. 2. Heat denaturation of purified bovine erythrocyte Cu2Zn2Superoxide dismutase. (O) SOD prepared by treatment with chloroform/ethanol, (A) aqueously isolated enzyme and ( ) the same enzyme stored for three months as lyophilized powder at room temperature. When freshly prepared, all fractions of aqueously isolated superoxide dismutase showed the same behaviour as (A) regardless of the age of the animal and the isoelectric point. SOD-activity was estimated using the cytochrome c assay...

The Cu2Zn2Superoxide dismutase of bovine erythrocytes is a homodimer of 31,300 daltons and contains one g atom of both, copper and zinc per subunit. SOD s from fungi, plants and vertebrates have remarkable homologies in the amino acid sequence . The sequence of the bovine erythrocyte enzyme is summarized in Table 3... 

Superoxide dismutase (SOD) was discovered in the 1960s by McCord and Fridovich in bovine erythrocytes where they described its functions as an enzyme which catalyzes the dismutation of superoxide radicals (Oj + O2 + 2H 02 + HjOj) (McCord and Fridovich 1969). Since the discovery of SOD, there are four isofoims of the enzyme which have been identified and they include Ni-SOD (cytoplasmic), Mn-SOD (mitochondrial), Cu/Zn-SOD (cytoplasmic), and EC-SOD (extracellular) (Mates and Sanchez-Jimenez 1999). The product of the dismutation of two superoxide anions carried out by SOD is the ROS, hydrogen peroxide, therefore biologically this enzyme is often coupled with antioxidants that remove hydrogen peroxide such as catalase and glutathione peroxidase. 

Adriamycin hydrochloride (ADM) was kindly supplied by Kyowa Hakko Co. (Japan) and used as received. 7-DADMN was derived from ADM, other quinoid compounds used are listed in Table 1. All of them except 9 were commercially available and purified by sublimation and recrystalliza-tion 3. Compound 9 was synthesized by alkylation of 1 with dimethyl sulfate. After being extracted with ether, it was recrystallized from methanol. The elemental analysis data for all the samples agreed well with the calculated values. Superoxide dismutase (SOD) (from bovine erythrocytes) was purchased from Sigma and used as received. Other chemicals were of reagent grade quality. 

SOD isolated from bovine liver or erythrocytes has been used medically as an anti-inflammatory agent. Human SOD has also been expressed in several recombinant systems, and is currently being evaluated to assess its ability to prevent tissue damage induced by exposure to excessively oxygen-rich blood. 

Since the discovery of superoxide dismutase (SOD) by McCord and Fridovich (Ml9), increasing numbers of papers have been published concerning the structural and functional aspects of this enzyme. Historically, a variety of SODs were reported as copper-containing proteins hemocuprein from bovine blood (M2), hepatocuprein from horse liver (M28), cerebrocuprein from human brain (P10), and ery throcupurein from human and beef erythrocytes (M9). Carrico and Deutsch (C2) gave the name cytocuprein to these proteins because the cupreins from various organs were found to be essentially identical. Cytocuprein was found to... 

BSOD, Bovine SOD from erythrocytes HSOD, human SOD. 

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