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Acknowledgments This rcscarch was supported by the University of Wisconsin College of Agriculture and Life Sciences and Graduate School and by a National Institutes of Health Biomedical Research grant administered by the Graduatc School. Contribution 3111 from the Department of Genetics.

[25] Analysis of E x t r a c e l l u l a r S u p e r o x i d e D i s m u t a s e in T i s s u e H o m o g e n a t e s a n d E x t r a c e l l u l a r Fluids B y STEFAN L . MARKLUND

Introduction Extracellular superoxide dismutase (EC 1.15.1. l, EC-SOD) is a secretory, tetrameric, copper- and zinc-containing glycoprotein with a subunit molecular weight of about 30,000.1'2 EC-SOD is the major SOD isoenzyme in extracellular fluids, such as plasma, lymph,a and synovial fluid: Although EC-SOD is the least predominant SOD isoenzyme in tissues, 90-99% of the EC-SOD in the body of mammals is located in the extravascular space of tissues) ,6 A prominent feature of EC-SOD is its affinity for heparin. On chromatography on heparin-Sepharose, plasma EC-SOD from man, 7 pig, cat, mouse, guinea pig, and rabbit s can be divided into at least three fractions: (A) a fraction without weak heparin affinity, (B) a fraction with weak heparin affinity, and (C) a fraction which elutes relatively late in a NaCl gradient. EC-SOD from tissues is mainly composed of forms with high heparin affinity (S. L. Marklund, unpublished data). In rat plasma, however, only fractions A and B can be demonstrated,a The binding to heparin is of electrostatic nature. 9 Since EC-SOD carries a net negative charge at neutral pH, the binding to the strongly negatively charged heparin molei S. L. Marklund, Proc. Natl. Acad. Sci. U.S.A. 79, 7634 (1982). 2 L. Tibell, K. Hjalmarsson, T. Edlund, G. Skogman, A. Engstr6m, and S. L. Markhind, Proc. Natl. Acad. Sci. U.S.A. 84, 6634 (1987). 3 S. L. Marldund, E. Holme, and L. Hellner, Clin. Chim. Acta 126, 41 (1982). 4 S. L. Marldund, A. Bjelle, and L.-G. Elmqvist, Ann. Rheum. Dis. 45, 847 (1986). 5 S. L. Marklund, J. Clin. Invest. 74, 1398 (1984). 6 S. L. Marklund, Biochem. J. 222, 649 (1984). 7 K. Karlsson and S. L. Marklund, Biochem. J. 242, 55 (1987). s K. Karlsson and S. L. Marklund, 8iochem. J. 255, 223 (1988). 9 K. Karlsson, U. Lindahl, and S. L. Marklund, Biochem. J. 256, 29 (1988).

METHODS IN ENZYMOLOOY, VOL. 186

Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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cule must be mediated by a cluster of positively charged amino acid residues in the enzyme. Such a cluster occurs in the very hydrophilic carboxy-terminal end of EC-SOD C, which contains three lysines and six arginine residues among the last 20 amino acids. 1° The differences between EC-SOD A, B, and C probably reside in this region. The in oioo correlate of the heparin affinity is apparently binding to heparan sulfate proteoglycan in the glycocalyx of cell surfaces. 7-9,11 The middle portion of the EC-SOD amino acid sequence shows a strong similarity with that part of the Cu,Zn-SOD sequence which defines the active site.l° The degree of homology in this portion between human EC-SOD and Cu,Zn-SODs from man, pig, cow, horse, swordfish, fruit fly, spinach, and bakers' yeast does not differ significantly. Only the Cu,Zn-SOD from Photobacterium leiognathi was clearly less homologous. 1°The data indicate that the EC-SODs have evolved from the Cu,ZnSODs or that they have a common ancestry. The divergence may have occurred before the evolution of plants and fungi. The EC-SODs may thus be widely distributed among higher phyla. So far EC-SODs have only been sought and found in mammals, 3,5,6,8birds, and fish (S. L. Marklund, unpublished data). For continued research on the functional role of the EC-SODs, their distribution in the body, as well as their phylogenetic occurrence, methods allowing their specific analysis are of the essence. The present chapter deals with methods aiding in distinguishing between EC-SOD and other SOD isoenzymes. EC-SOD is, like the CuZn SODs but unlike Mn-SODs and Fe-SODs, very sensitive to inhibition by cyanide. The problem is thus reduced to distinguishing between the cyanide-sensitive isoenzymes EC-SOD and Cu,Zn-SOD. We have so far not found any distinguishing inhibitor. In accord with the similarities in active site structure, 1° both isoenzymes are inhibited by cyanide, azide, H202, diethyl dithiocarbamate, 12 and the arginine-specific reagent phenylglyoxal. t3 Availability of polyclonal or monoclonal antibodies toward ECSOD and/or Cu,Zn-SOD solves the problem of distinction within a certain species, since we have not found any antigenic cross-reactivity between the isoenzymes.12 However, our antihuman EC-SOD antibodies produced so far have reacted poorly or not at all with EC-SOD from other mammals. More generally applicable procedures for distinction must be based on physical differences between the isoenzymes. Below are described methods employing differences in size and affinity for lectins and heparin. ~o K. Hjalmarsson, S. L. Marklund,/~. Engstrfm, and T. Edlund, Proc. Natl. Acad. Sci. U.S.A. 84, 6340 (1987). ii K. Karlsson and S. L. Marklund, J. Clin. Invest. 82, 762 (1988). 12 S. L. Marklund, Biochem. J. 220, 269 (1984). 13 T. Adachi and S. L. Marklund, J. Biol. Chem. 264, 8537 (1989).

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Experimental Procedures

Preparation of Samples Tissues are homogenized with an Ultra-Turrax (or similar apparatus) in 10 volumes of ice-cold 50 m M potassium phosphate, pH 7.4, with 0.3 M KBr (chaotropic salt that increases extraction of EC-SOD severalfoldS), 3 mM diethylenetriaminepentaacetic acid, 100 klU/ml aprotinin, and 0.5 mM phenylmethylsulfonyl fluoride (the latter three additions inhibit proteases). The tissue extract may then be subjected to ultrasonication followed by centrifugation (20,000 g, 20 min). The supernatants are then used for the separations. For subsequent separation on Con A-Sepharose or with gel chromatography, samples can be processed fresh or thawed after storage below - 7 0 °. The affinity for heparin is, however, very sensitive to treatment of the samples and is easily lost, probably owing to proteolysis of the carboxy-terminal end, which is responsible for heparin binding. To avoid this, tissues should preferably be processed fresh. If the tissues are allowed to thaw after freezing, we have noted a loss of high 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 mechanically pulverized at -196 ° (liquid N/) followed by addition of ice-cold extraction buffer and subsequent sonication without loss of heparin affinity. In the final extract, the antiproteolytic measures seem to prevent further degradation. The extracts can be kept at - 7 0 °" The stability problem is apparently not so great in extracellular fluids. The plasma EC-SOD heparin affinity pattern was not influenced by freezing and thawing or by a 3-day storage in a refrigerator. 7 Apparently the large amounts of protein and the numerous antiproteases in plasma confer protection. EDTA (or citrate) but not heparin should be used as anticoagulant, since heparin might interfere with the heparin-Sepharose procedure and also induces a large increase in the apparent molecular mass of ECSOD C. a

Analysis of SOD Activity The EC-SOD activity is low both in extracellular fluids and in tissue extracts. The amount of EC-SOD in the fractions collected in the suggested procedures is consequently very low and varies between about 4 and I00 ng/ml. Concentrations this small cannot be determined with most common SOD assays. Highly sensitive SOD assays are necessary. We only use the direct spectrophotometric assay with 02 ~ obtained from

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KO2.14,15 A unit corresponds to 4-9 ng Cu,Zn-SOD and EC-SOD, and the assay is 40 times more sensitive than the xanthine oxidase-cytochrome c procedure.~6 Separation by Gel Chromatography

The principle behind gel chromatography is that most EC-SODs have an apparent molecular weight of around 150,000 on gel chromatography, whereas Cu,Zn-SOD elutes at a position corresponding to 30,000. ECSOD in plasma from man, cat, pig, sheep, mouse, rabbit, guinea pig, and rat was very easily distinguished from the small amounts of Cu,Zn-SOD that occur. 3,8 The rat plasma EC-SOD elutes at an apparent molecular weight of 90,000. 8 Whether this is due to, for example, smaller subunits or to a dimeric state is not yet known. Such atypical molecular weights might also occur in the EC-SODs from other phyla. In tissue extracts EC-SOD is a minor isoenzyme, but it is still usually possible to assess the amount of EC-SOD from the gel chromatography pattern. 5,6 This was not possible in rat tissue homogenates, however, where the EC-SOD peak was hidden by the large Cu,Zn-SOD and Mn-SOD peaks. 6 Procedure. Any gel chromatography system with good resolution in the range 150-30 kDa can probably be used. We use the following procedure. The sample (1-5 ml) is applied to a column (1.6 × 90 cm) of Sephacryl S-300 (Pharmacia LKB Biotechnology, Bromma, Sweden), eluted at 20 ml/hr with 10 mM potassium phosphate (pH 7.4), 0.15 M NaCI, and collected in 3-ml fractions. The absorbance at 280 nm and the SOD activity are determined in the collected fractions. Separation on Con 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, l,z Chromatography of samples on concanavalin Asubstituted Sepharose (Con A-Sepharose, Pharmacia) has proved to be a useful procedure for distinguishing EC-SOD from the 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 varies mostly between 70 and 90%. Procedure. The chromatography is executed manually in a stepwise fashion. The tissue extract (1-2 ml) is applied to a l-ml Con A-Sepharose t4 S. L. Marklund,J. Biol. Chem. 251, 7504 (1976). 15S. L. Marklund, in "Handbook of Methods for Oxygen Radical Research" (R. Greenwald, ed.), p. 249. CRC Press, Boca Raton, Florida, 1985. 16j. M. McCord and I. Fridovich,J. Biol. Chem. 244, 6049 (1969).

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column equilibrated with 50 m M N a - H E P E S (pH 7.0), 0.25 M NaC1. The sample is applied in 0.5-ml portions with 5-min intervals between applications. After 5 min, 3 ml equilibration buffer is added. The eluting fluid from the tissue extract and buffer additions is collected and contains the SOD activity which lacks Con A affinity. The column is then washed with 10 ml equilibration buffer. EC-SOD is finally eluted with 5 ml of 0.5 M amethylmannoside dissolved in equilibration buffer added in 1-ml portions with 5-min intervals. The column is regenerated with 5 ml of 0.5 M amethylmannoside followed by 10 ml of equilibration buffer. Chromatography on Heparin-Sepharose Procedure. The chromatography is carried out on a 2-ml HeparinSepharose column equilibrated with 15 mM sodium cacodylate (pH 6.5), 50 mM NaCI, 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. 7 The samples should be dialyzed against the elution 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 man, pig, cat, mouse, rabbit, and guinea pig plasma the B fractions eluted between 0.17 and 0.30 M NaC1 and the C fractions between 0.42 and 0.62 M NaCI. 7,8 In all these species Cu,ZnSOD and Mn-SOD eluted without binding, together with EC-SOD A. To distinguish between EC-SOD A and the other isoenzymes in the nonbinding fraction, the Con A-Sepharose procedure 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 is given by EC-SOD. The strongly negatively charged heparin-Sepharose gel will also function as a cation-exchange chromatography column. The net charge of Cu,Zn-SOD and Mn-SOD from other taxa might be such that they bind to the heparin-Sepharose. General Comments Demonstration and quantification o f EC-SOD using the procedures outlined here should be possible in samples from most mammalian species. Analysis in other phyla is more uncertain, since we have as yet no

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generally applicable knowledge concerning molecular weights, glycosylation, and heparin affinity of the secretory EC-SODs. Combination of the procedures should aid in identifying a SOD fraction as an EC-SOD. For example, a cyanide-sensitive SOD fraction with high heparin affinity that binds to concanavalin A and/or has a high molecular weight is likely to be an EC-SOD. Better procedures might emerge as EC-SODs from a wider phylogenetic spectrum are isolated and characterized.

[26] O x i d a t i v e R e a c t i o n s o f H e m o g l o b i n B y CHRISTINE C. WINTERBOURN

Introduction Hemoglobin readily undergoes one-electron oxidations and reductions, and it can act as a source or sink of free radicals. Autoxidation of the heme groups produces O2- and, indirectly, H202.1,2Hemoglobin also interacts with redox-active xenobiotics and metabolites, forming the xenobiotic radical and initiating a series of reactions that generate other radicals and oxidant species and often result in oxidative denaturation of the hemoglobin. 3 In the red cell, the outcome can be hemoglobin precipitation as Heinz bodies and oxidative damage to other cellular constituents. Production of 02- and other radicals can be detected by conventional techniques (e.g., superoxide dismutase-inhibitable cytochrome c reduction or electron spin resonance with or without the use of a spin trap). Care must be taken with interpretation, however, because intermediates of hemoglobin oxidation can sometimes participate in the detection reaction. This chapter focuses on the measurement of oxidant-mediated changes to hemoglobin. First, I shall define the different oxidation products of hemoglobin and briefly describe general mechanisms for their production. Oxidation of oxyhemoglobin (oxyHb) 4 gives 02- and methemoglobin (metHb). If the globin structure is destabilized, metHb can convert to hemichrome, in which either the distal histidine or an external i H. P. Misra and I. Fridovich, J. Biol. Chem. 247, 6960 (1972). 2 C. C. Winterbourn, B. M. McGrath, and R. W. Carrell, Biochem. J. 155, 493 (1976). 3 C. C. Winterbourn, Environ. Health Perspect. 64, 32 (1985). 4 Abbreviations: oxyHb, oxyhemoglobin; metHb, methemoglobin; ferrylHb, ferrylhemoglobin; DTNP, 2,2'-dithiobis(5-nitropyridine); SDS-PAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.

METHODS IN ENZYMOLOGY, VOL. 186

Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Analysis of extracellular superoxide dismutase in tissue homogenates and extracellular fluids.

260 ASSAY OF F O R M A T I O N OR R E M O V A L OF O X Y G E N RADICALS [25] Acknowledgments This rcscarch was supported by the University of Wisco...
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