74 SUPEROXIDE REACTIONS AND MECHANISMS [7]

o n kcat/K m. Replacement of Gln-143 causes a much greater decrease in catalytic activity, by two to three orders of magnitude, and causes significant changes to the redox potential as well. During catalysis, MnSOD is inhibited by a peroxide complex of the metal in the active site, different from the inhibition of FeSOD and Cu,ZnSOD by Fenton chemistry. Site-specific mutagenesis of active-site residues alters the extent of product inhibition of MnSOD as well, indicating that this is not only a property of the metal. The replacement of Trp-161 with phenylalanine results in a variant that is completely blocked in catalysis by product inhibition.

[7] Extracellular Superoxide Dismutase

B y STEFAN L. MARKLUND

I n t r o d u c t i o n

Mammalian extracellular superoxide dismutase (EC 1.15.1.1, EC-SOD) is a secreted, tetrameric, copper- and zinc-containing glycoprotein with a molecular mass of the subunit protein of about 25,000 Da. 1-3 EC-SOD is the major SOD isoenzyme in extracellular fluids such as plasma, lymph, 4 and synovial fluid. 5 It also occurs in cerebrospinal fluid, 6 and seminal plasma, 7 but here the normally cytosolic Cu,ZnSOD shows higher activity. In mammals 90-99% of the EC-SOD is located in the tissues. Most of this tissue enzyme is probably bound to heparan sulfate proteoglycans on cell surfaces, in basal membranes, and in the connective tissue matrix. 8-1° However, in some cases, for example, placenta, 11 and lung during development, 12 in mouse brain, 13 and mouse aorta (S. L. Marklund, unpublished data, 2001), a large portion of the enzyme stains intracellularly. It may be stored in

1 S. L. Marklund, Proc. Natl. Acad. Sci. U.S.A. 79, 7634 (1982). 2 L. Tibell, K. Hjalmarsson, T. Edlund, G. Skogman,/~. Engstr6m, and S. L. Marklund, Proc. Natl.

Acad. Sci. U.S.A. 84, 6634 (1987). 3 L. M. Carlsson, S. L. Marklund, and T. Edlund, Proc. Natl. Acad. Sci. U.S.A. 93, 5219 (1996). 4 S. L. Marklund, E. Holme, and L. Hellner, Clin. Chim. Acta 126, 41 (1982). 5 S. L. Marklund, A. Bjelle, and L.-G. Elmqvist, Ann. Rheum. Dis. 45, 847 (1986). 6 j. Jacobsson, E A. Jonsson, E M. Andersen, L. Forsgren, and S. L. Marklund, Brain 124, 1461

(20Ol). 7 R. Peeker, L. Abramsson, and S. L. Marklund, Hum. Mol. Reprod. 3, 1061 (1997). g K. Karlsson and S. L. Marklund, Lab. Invest. 60, 659 (1989). 9 K. Karlsson, J. Sandstrtim, A. Edlund, T. Edlund, and S. L. Marldund, Free Radic. Biol. Med. 14,

185 (1993). t0 K. Karlsson, J. Sandstr6m, A. Edlund, and S. L. Marklund, Lab. Invest. 70, 705 (1994). I l K. A. Boggess, H. H. Kay, J. D. Crapo, W. F. Moore, H. B. Suliman, and T. D. Oury, Am. J. Obstet,

Gynecol. 183, 199 (2000).

Copyright 2002, Elsevier Science (USA). All rights reserved.

METHODS IN ENZYMOLOGY, VOL 349 0076-6879/02 $35.00

[7] EXTRACELLULAR SUPEROXIDE DISMUTASE 75

secretory vesicles, and whether it there contributes to protection against the super- oxide radical is unknown. Consequently, the total content of EC-SOD measured in a tissue extract does not necessarily reflect the SOD activity in the extracellular space.

A distinguishing feature of EC-SOD is its affinity for heparin and other sulfated glycosaminoglycans, primarily heparan sulfate proteoglycan, 14 which determines the distribution and retention of the enzyme in vivo. 8-1° The affinity is conferred by a carboxy-terminally located positively charged heparin-binding domain. 15 On chromatography on heparin-Sepharose, plasma EC-SOD from most mammals is divided into at least three fractions: A, which lacks affinity; B, which shows inter- mediate affinity; and C, which has high affinity. 16,17 The fractions with reduced affinity are composed of heterotetramers containing subunits with proteolyti- cally truncated carboxy-terminal ends. 15,18 Tissue EC-SOD is mainly composed of homotetrameric high-affinity type C. is The rat lacks the high-affinity type C. 17 The heparin-binding domain of the rat EC-SOD is identical to those of the mouse and human enzymes. However, owing to a difference in one criti- cal amino acid residue in a subunit interaction area, rat EC-SOD is dimeric. 3 High heparin affinity thus requires the cooperative action of four heparin-binding domains.

The middle portion of the EC-SOD sequence shows high similarity with that part of the CuZn-SOD sequence that defines the active site. All the ligands to the prosthetic metals as well as several others for the function of important residues can be identified in EC-SOD ? The spectral properties of EC-SOD are similar to those of the Cu,ZnSODs,19and the enzyme behaves like Cu,ZnSOD in the Tsuchihashi chloroform-methanol procedure.l The consequence of this is that all the inhibitors and treatments commonly used to identify the cyto- solic Cu,ZnSODs, affect EC-SOD almost identically. Thus, EC-SOD is reversibly inhibited by cyanide and azide, and inactivated by diethyldithiocarbamate, phenyl- glyoxal, and hydrogen peroxide. 2° In general, the EC-SODs are more sensitive to these treatments than the Cu,ZnSODs. 2° We have so far failed to identify any inhibitor that efficiently distinguishes between the mammalian copper-containing SODs; EC-SOD and Cu,ZnSOD.

le E. Nozik-Grayk, C. S. Dieterle, C. A. Piantadosi, J. J. Enghild, and T. D. Oury, Am. J. Physiol. Lung. Cell. Mol. Physiol. 279, L997 (2000).

13 T. D. Oury, J. P. Card, and E. Klann, Brain Res. 850, 96 (1999). 14 K. Karlsson, U. Lindahl, and S. L. Marklund, Biochem. J. 256, 29 (1988). 15 j. Sandstrtim, L. Carlsson, S. L. Marklund, and T. Edlund, J. Biol. Chem. 267, 18205 (1992). 16 K. Karlsson and S. L. Marklund, Biochem. J. 242, 55 (1987). 17 K. Karlsson and S. L. Marklund, Biochem. Z 255, 223 (1988). 18 j. Sandstr6m, K. Karlsson, T. Edlund, and S. L. Marklund, Biochem. J. 294, 853 (1993). 19 L. Tibell, R. Aasa, and S. L. Marklund, Arch. Biochem. Biophys. 304, 429 (1993). 20 S. L. Marklund, Biochem. J. 220, 269 (1984).

76 SUPEROXIDE REACTIONS AND MECHANISMS [7]

Here we describe some methods that can be used to probe the physical proper- ties of EC-SOD and that can aid in distinguishing the enzyme from the Cu,ZnSODs.

E x p e r i m e n t a l P r o c e d u r e s

Preparation of Samples

Tissues are homogenized with a mixer [Ultra-Turrax (IKA Labortechnik, Staufen, Germany) or similar apparatus] in 10-25 volumes of ice-cold potassium phosphate, pH 7.4, with 0.3 M KBr (chaotropic salt that may increase the extrac- tion severalfold from some tissues21), 3 mM diethylenetriaminepentaacetic acid, 100 klU of aprotinin, and 0.5 mM phenylmethylsulfonyl fluoride [the latter three additions to inhibit proteases; commercial cocktails such as Complete (Boehringer, Mannheim, Germany) can also be used]. The tissue extract may then be subjected to ultrasonication followed by centrifugation (20,000g, 20 min, 4°). The super- natants are used for the separations.

For subsequent separation on concanavalin A (ConA)-Sepharose, by gel chro- matography, or with immobilized antibodies, samples can be processed fresh or after storage below - 7 0 °. The affinity for heparin is, however, sensitive to treatment of the samples and is easily lost owing to proteolytic truncation of the carboxy- terminal heparin-binding domain. 18 To avoid this, tissues should preferably be processed fresh. If the tissues are allowed to thaw after freezing, we have noted a loss of heparin affinity. We interpret the effect to be due to proteolytic enzymes released by freezing-induced cellular and subcellular rupture. Frozen tissues can, however, be pulverized at liquid N2 temperature, followed by addition of ice-cold extraction buffer and subsequent sonication without loss of heparin affinity. 18 In the final extract, the antiproteolytic measures seem to prevent further degradation.

The stability problem is apparently not so great in plasma. The plasma EC-SOD heparin affinity pattern was not influenced by freezing and thawing or by a 3-day storage in a refrigerator. ~6 Apparently the large amounts of protein and the numer- ous antiproteases in plasma confer protection. EDTA (or citrate) should be used as anticoagulant. Heparin interferes with the heparin-Sepharose procedure and also induces a large increase in the apparent molecular mass of EC-SOD fraction C.17

Highly Diluted Samples

Sometimes little material is available, which is why larger dilutions than sug- gested above must be used. This will lead to markedly reduced recovery of EC-SOD by the various procedures suggested, probably because of unspecific adsorption of the enzyme to surfaces. We have found that addition of 0.2% bovine serum albumin to the extracts and media used for the separations generally prevents the problem.

21 S. L. Marklund, J. Clin. Invest. 74, 1398 (1984).

[7] EXTRACELLULAR SUPEROXIDE DISMUTASE 77

Analysis of Superoxide Dismutase Activity

The EC-SOD activity is low both in extracellular fluids and in most tissue extracts. The amount of EC-SOD in the fractions collected by the suggested pro- cedures is consequently low and varies between about 4 and 200 ng/ml. The cor- responding activities are difficult to analyze with the more common SOD assays. Highly sensitive assays are necessary, and we use only the direct spectrophoto- metric assay with superoxide obtained from KO2 .22,23

Separation by Gel Chromatography

The principle behind the procedure is that most EC-SODs show an apparent molecular mass of about 150,000 Da on gel chromatography, whereas Cu,ZnSOD elutes at a position corresponding to 30,000 Da. The high apparent molecular mass is dependent on the hydrodynamic effects of the single carbohydrate chains on the subunits; mutant nonglycosylated human EC-SOD elutes close to the expected molecular mass of 97,000 Da. 24 EC-SOD in plasma from human, cat, pig, sheep, mouse, rabbit, guinea pig, and rat can easily be separated from the relatively small Cu,ZnSOD peaks. 17 The rat EC-SOD, which is dimeric, 3 elutes at an apparent molecular mass of 90,000 Da. Such atypical molecular masses might also occur in EC-SODs from other phyla. In tissue extracts EC-SOD is mostly a minor isoen- zyme, but it is still usually possible to assess the amount of EC-SOD from the gel chromatography pattern. This was not possible in rat tissue homogenates, how- ever, where the EC-SOD peak was hidden behind the large Cu,ZnSOD and MnSOD peaks. 25

Procedure. Any gel chromatography system with good resolution in the range of 30-150 kDa can be used. We typically use the following procedure. The sample (1-5 ml) is applied to a column (1.6 x 90 cm) of Sephacryl S-300 (AP Biotech, Uppsala, Sweden), eluted at 20 ml/hr with 10 mM potassium phosphate (pH 7.4)- 0.15 M NaC1, and collected in 3-ml fractions. The absorbance at 280 nm and the SOD activity are determined in collected fractions.

Separation on Concanavalin A-Sepharose

EC-SOD is, unlike the other SOD isoenzymes, a glycoprotein and has been found to bind to lectins such as concanavalin A, lentil lectin, and wheat germ lectin. 1,2 Chromatography of samples on concanavalin A-substituted Sepharose

22 S. L. Marklund, J. BioL Chem. 251, 7504 (1976). 23 S. L. Marklund, in "Handbook of Methods for Oxygen Radical Research" (R. Greenwald, ed.),

p. 249. CRC Press, Boca Raton, Florida, 1985. 24 A. Edlund, T. Edlund, K. Hjalmarsson, S. L. Marklund, J. Sandstrrm, M. Strrmkvist, and L. Tibell,

Biochem. J. 288, 451 (1992). 25 S. L. Marklund, Biochem. J. 222, 649 (1984).

78 SUPEROXIDE REACTIONS AND MECHANISMS [7]

(ConA-Sepharose; AP Biotech) has proved to be a useful and reliable procedure for distinguishing EC-SOD from other SOD isoenzymes. The EC-SOD is bound and can then be eluted with a-methylmannoside. The recovery of EC-SOD in the suggested procedure is about 70-80%. As tested with pure enzyme or by analysis of extracts and collected fractions with an enzyme-linked immunosorbent assay (ELISA) for EC-SOD, the recovery from human extracts is regularly close to 75%. We always compensate our results for that.

Procedure. The chromatography is carried out manually in a stepwise fashion. The tissue extract (1-2 ml) is applied to a 1-ml ConA-Sepharose column equi- librated with 50 mM Na-HEPES (pH 7.0)-0.25M NaC1. The sample is applied in 0.5-ml portions at 5-min intervals. After 5 min, 3 ml of equilibration buffer is added. The eluting fluid from the tissue extract and buffer additions is collected and contains the SOD activity that lacks ConA affinity. The column is then washed with 10 ml of equilibration buffer. EC-SOD is finally eluted with 5 ml of 0.5 M a-methylmannoside added in 1-ml portions at 5-min intervals. The column is re- generated with 5 ml of ot-methylmannoside followed by 10 ml of equilibration buffer.

Chromatography on Heparin-Sepharose

Procedure. The chromatography is carried out on a 2-ml heparin-Sepharose column equilibrated with 15 mM sodium cacodylate, pH 6.5, with 50 mM NaC1, eluted at 5 ml/hr. The tissue extract (1-10 ml) or plasma (up to 2 ml) is applied. Many plasma proteins bind to heparin, and if more than 2 ml is applied there is a risk of column saturation. 16 The samples should be dialyzed against the equilibration buffer. After application of samples, the column is eluted with 15 ml of the buffer. Thereafter, bound proteins are eluted with a linear gradient of NaC1 in the buffer (0-1 M; total volume, 50 ml). The eluent is collected in 1.5-ml fractions, and the SOD activity and absorbance at 280 nm are determined.

By definition EC-SOD A is the fraction that elutes without binding, B is the fraction that elutes early in the gradient, and C is the fraction that elutes relatively late. In human, cat, pig, mouse, rabbit, and guinea pig plasma, the B fractions eluted between 0.17 and 0.30 M NaC1, and the C fractions eluted between 0.42 and 0.62 M NaCl. 16,17 In all these species Cu,ZnSOD and MnSOD eluted without binding, together with EC-SOD A. To distinguish between EC-SOD A and the other isoenzymes in the nonbinding fraction, the ConA-Sepharose procedure or immobilized antibodies can be used. All SOD activity in the gradient in these species was given by EC-SOD. Note, however, that it cannot be taken for granted that all SOD activity in the gradient represents EC-SOD. The strongly negatively charged heparin-Sepharose gel will also function as a cation-exchange chroma- tography column. The net charge of Cu,ZnSODs and MnSODs from other taxa might be such that they bind to the column.

[7] EXTRACELLULAR SUPEROXIDE DISMUTASE 79

Analyses with Antibodies

Despite some sequence similarities we have so far not found any antigenic cross-reactivity between human EC-SOD and Cu,ZnSODs from a few mammalian species. Apparently immunological methods can safely be used for specific iden- tification of the two isoenzymes.

Westem blots and ELISAs function well for both, and are standard methods. Regarding immunohistochemistry we generally find the staining for Cu,ZnSODs to be more robust than that for EC-SOD. The antigenic reactivity of EC-SOD appears sensitive to strongly denaturing or cross-linking fixatives such as formaldehyde and glutaraldehyde, and better staining is often seen with frozen sections or with alcohol fixation. However, proteolytic digestion (proteinase 1; Ventana, Tucson, AZ) has markedly improved the staining of formalin-fixed paraffin-embedded samples.

A convenient means of distinguishing between EC-SOD and Cu,ZnSOD in extracts is to use antibodies against the enzymes immobilized on particles such as Sepharose. The extracts are then incubated with the immobilized antibodies or control particles, and after centrifugation the (cyanide-sensitive) SOD activities of the supernatants are analyzed. The lost activity corresponds to the SOD isoenzyme against which the antibody is directed, and the remaining activity corresponds to the other isoenzyme. At this time, commercial antibodies versus EC-SOD are not available, whereas antibodies versus Cu,ZnSOD are easier to find. Using antibodies versus Cu,ZnSOD, distinction between the activities of the two isoenzymes can be achieved.

Procedure. The antibody is incubated with CNBr-activated Sepharose (AP Biotech) at a concentration of 10 mg of protein per milliliter of swollen gel, fol- lowed by blocking and washing according to the instructions of the manufacturer. The capacity of the immobilized antibody is then tested by incubation of the gel with a solution containing SOD. Isolated enzyme can be used, or a tissue extract containing only the isoenzyme of interest. If, for example, an anti-Cu,ZnSOD antibody is to be tested, different dilutions of a hemolysate can be used. Typically we add 50/zl of antibody-gel to a series of tubes containing 1 ml of enzyme/tissue extract/hemolysate with stepwise increasing (doubling) concentrations. The tubes are gently shaken at 4 ° for a couple of hours or overnight. After centrifugation, the SOD activity is determined in the supernatants. The antibodies typically have bound about 100/zg of SOD per milliliter of wet gel. For the assay we never use more than 25% of the maximal capacity of the gel, which ensures almost complete adsorption of the SOD isoenzyme.

Tissue extracts diluted to suit the capacity of the gel (see above) in neutral buffer, for example, phosphate-buffered saline, are incubated with antibody-gel (typically 50 /zl of gel per milliliter of extract) or Sepharose 4B (blank). The suspensions are shaken at about 4 ° for 2hr or overnight. After centrifugation, the cyanide-sensitive SOD activities of the supernatants are analyzed.

80 SUPEROXIDE REACTIONS AND MECHANISMS [8]

General Comments

It should be possible to demonstrate and quantify EC-SOD in samples from most mammalian species, using the procedures outlined here. Secreted copper- containing SODs have been found in several other phyla. Whether some or all of these are on the same evolutionary branch as the mammalian EC-SODs or represent separate branches has not yet been comprehensively analyzed. Even less is known about specific properties such as heparin binding, glycosylation, and tetrameric versus dimeric state. The present procedures might help in probing for such properties.

[8] Prokaryotic Manganese Superoxide Dismutases B y JAMES W. WHITTAKER

I n t r o d u c t i o n

Manganese superoxide dismutases (MnSODs) 1-3 are the front-line antioxidant defense in many prokaryotes, protecting against oxidative challenges resulting from environmental or biological interactions. The enzymes are typically multi- mers of small subunits (,~23-kDa molecular mass), and are usually localized in the cytoplasm, 4 where they may be associated with DNA. 5'6 Enzymes from mesophilic organisms are generally homodimeric proteins, whereas thermophilic and hyper- thermophilic enzymes are often tetramers. 2 Each subunit contains a mononuclear manganese complex (Fig. 1) that forms the catalytic active site. The metal ion is re- dox active, shuttling between Mn(II) and Mn(III) oxidation states during turnover.

Superoxide dismutases are widespread among bacterial and archaeal life, oc- curring in both prokaryotic domains of the phylogenetic tree. 2 Even some organ- isms normally identified as strict anaerobes have been found to contain a superoxide dismutase, and the exceptional aerobes that lack the enzyme (i.e., the lactic acid bacteria) generally have a fermentative metabolism and may contain manganese salts that mimic SOD activity. 7 The following sections provide a basic guide to the properties of the prokaryotic MnSODs.

1 j. M. McCord, New Horiz. 1, 70 (1993). 2 I. Fridovich, J. Biol. Chem. 272, 18515 (1997). 3 j. W. Whittaker, Metals Biol. Syst. 37, 587 (2000). 4 H. M. Steinman, L. Weinstein, and M. Brenowitz, J. Biol. Chem. 269, 28629 (1994). 5 K. A. Hopkin, M. A. Papazian, and H. M. Steinman, J. Biol. Chem. 267, 24253 (1992). 6 R. A. Edwards, H. M. Baker, M. M. Whittaker, J. W. Whittaker, G. B. Jameson, and E. N. Baker,

J. Biol. Inorg. Chem. 3, 161 (1998).

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