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In this view the difference between the green and the pink complexes is that the former contains oxidized Desferal while the latter contains intact Desferal. Oxidation of one of its hydroxamate ligand groups should decrease the affinity of the Desferal for Mn(III), and, as expected, the green complex was less stable to EDTA than was the pink complex. Desferal is known to act as an electron donor, and the nitroxide product of the univalent oxidation of one of its hydroxamate groups has been studied by electron paramagnetic resonance. ~° Since the pink complex was prepared by reacting 1.0 mol ascorbate per mole of Desferal with a 25% molar excess of MnO2, it must be presumed to contain dehydroascorbate. Dehydroascorbate at 250 /xM dd not inhibit the reduction of cytochrome c by the flux of 02- generated by the action of xanthine oxidase on 10 mM CH3CHO. It follows that this concentration of dehydroascorbate did not exhibit any detectable ability to intercept 02- and that the very much lower concentration (4/zM) present during assays of the pink complex would not interfere with assays of the activity of that complex. The greater stability and catalytic activity of the pink complex suggest that it may be a more useful mimic of SOD activity than the green complex. This remains to be explored in vivo. Acknowledgments This work was supported by research grants from the National Science Foundation, the Council for Tobacco Research--USA, Inc., and the National Institutes of Health. 10 K. M. Morehouse, W. D. Flitter, and R. P. Mason, FEBS Lett. 222, 246 (1987).
[24] Purification of E x o 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 s
By KENNETH D. MUNKRES Introduction
Although superoxide dismutases (SODs, EC 1.15.1.1) have been intensively investigated for many years, the existence of exocellular forms of the enzyme was recognized only in the 1980s. The existence of exocellular forms of the enzyme (EC-SODs) in bovine milk was discovered almost simultaneously in three laboratories around 1975. ~ Subsequently, 1 M. Korycka-Dahl, T. Richardson, and C. L. Hicks, J. Food Prot. 42, 867 (1979).
METHODS IN ENZYMOLOGY, VOL. 186
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Marklund and associates 2-5 reported a series of investigations of EC-SODs from human and animal tissues. Marklund 2 purified human lung EC-SOD on a large scale with low yield by a lengthy series of ion-exchange and affinity chromatographic steps. Subsequently, the human EC-SOD structural gene was cloned, and the enzyme was synthesized in large quantities by genetic engineering techniques. 4,5 The complete amino acid sequence of the polypeptide subunit and signal peptide of the enzyme was deduced from the nucleotide sequence of the gene. Comparison of sequence similarities indicated that the exo- and endocellular SOD isozymes arose from a common ancestral enzyme before the evolution of fungi and plants; hence, one may infer a broad phylogenetic distribution of the exoenzyme. The results of this investigation support that inference. Facile procedures for large-scale purification and crystallization were invented and applied to bovine milk and blood serum, wheat germ, yeast, and bacteria. The enzyme was also partially purified from Neurospora. 6 The widespread phylogenetic distribution of the enzyme indicates that it plays an essential but yet unknown biological role. Knowledge of its biological role may be significant for application of the engineered human enzyme in proposed clinical therapy of human diseases mediated by free radicals. Our laboratory is investigating the genetic regulation and biological function of EC-SOD. EC-SOD-deficient mutants have been isolated in N e u r o s p o r a and yeast. 6 The aim of this study was to develop the technology to conduct comparative EC-SOD therapy of the physiological "diseases" of the mutants. Human EC-SOD is a tetrameric glycoprotein whose molecular weight is 135,000. 2 The present purification procedures are designed to select proteins of this nature. Materials Defatted, pasteurized dry milk (Carnation Co., Los Angeles, CA), natural (untoasted) wheat germ (Hodgson Mill Enterprise, Gainsville, MO), and Red Star Quick-Rise dried bakers' yeast in vacuo (Universal Foods Corp., Milwaukee, WI) were obtained from local grocers. Lyophi-
2 S. L. Marklund, Proc. Natl. Acad. Sci. U.S.A. 79, 7634 (1982). 3 S. L. Marklund, Biochem. J. (London) 222, 649 (1984). 4 K. Hjalmarsson, S. L. Marklund, A,. Engstr6m, and T. Edlund, Proc. Natl. Acad. Sci. U.S.A. 84, 6340 (1987). 5 L. Tibell, K. Hjalmarsson, T. Edlund, G. Skogman, A. Engstr6m, and S. L. Marklund, Proc. Natl. Acad. Sci. U.S.A. 84, 6634 (1987). K. D. Munkres, unpublished (1988).
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lized cells of Bacillus subtilis (ATCC 6633) and Escherichia coli (strain W) and concanavalin A-agarose were obtained from Sigma Chemical Co. (St. Louis, MO). Sterile calf serum was from HyClone Laboratories (Logan, UT). Methods and Results
SOD Assay. The spectrophotometric method of Misra and Fridovich7 is used. The method is based on the inhibition by the enzyme of the spontaneous, alkali-catalyzed autoxidation of epinephrine to norepinephrine. Enzyme in 10-100/zl of distilled water diluent is added to 1 ml of the reaction mixture in a semimicrocuvette at 36°. The reaction rate is monitored in a Gilford recording spectrophotometer at 480 nm for 6 min. After a lag period of 1 to 3 min, the linear rate during the next 2 to 3 min is graphically determined. Plots of percent inhibition versus protein concentration are constructed with two or three points, and the concentration producing 50% inhibition (1 unit) is determined by interpolation. Usually the plots are linear from 20 to 80% inhibition. Rarely some samples exhibit linear plots that plateau at 40-60% inhibition. In those instances, a unit of activity is defined as that concentration producing half-maximal inhibition. Employing commercial bovine red blood cell SOD, we confirmed the observation of Misra 7,8 that the present assay method is about 2-fold more sensitive than the xanthine oxidase method of McCord and Fridovich. Ethanol mimics SOD: at least 1% inhibits the assay. Ethanol extracts should contain sufficient enzyme to permit its assay after dilution in water to noninhibitory ethanol concentrations. Tetraborate does not interfere with the assay. Protein Assay. The spectrophotometric method of Murphy and Kies 9 is used. Tetraborate Assay. The course of dialysis to remove tetraborate is monitored by a colorimetric assay. A sample of the dialysate (0.9 ml) is mixed with 0.1 ml of 1% silver nitrate and incubated at room temperature about 10 min or until the absorbancy at 500 nm becomes constant. The extinction coefficient of tetraborate (Emu, cm -j) is 0.255. The assay detects as little as 10-100/zg sodium tetraborate per milliliter. Extraction of Exocellular SOD. Survey of 16 strains of bakers', beer, and wine yeast indicated that Red Star Quick-Rise Bakers' yeast is an 7 H. P. Misra and I. Fridovich, J. Biol. Chem. 247, 3410 (1972). 8 H. P. Misra, in "CRC Handbook of Methods for Oxygen Radical Research" (R. A. Greenwald, ed.), p. 237. CRC Press, Boca Raton, Florida, 1985. 9 j. B. Murphy and M. W. Kies, Biochim. Biophys. Acta 45, 382 (1960).
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extraordinarily rich source of EC-SOD. 6 The EC-SOD activity of the yeast in vioo is determined by assay with washed cells diluted in water. The specific activity is about 0.89 units/mg dry cells. Two methods efficiently extract the enzyme, as indicated by either units extracted or units remaining with the cell. Method A uses 0.3 M KBr in 5 mM acetate buffer (pH 5.5); method B uses 80% ethanol. One gram of dry yeast is stirred 10-20 min with 10-20 volumes of extraction solvent at room temperature. Cells are removed by centrifugation at I0,000 g for 10 min. Two extractions yield at least 95% of the enzyme units initially present. Method A is preferred if we wish to subsequently break the cells and measure endocellular SOD activities. The exoenzyme may be concentrated and purified from the extract on a small scale by adjusting the extract to 80% ethanol followed by precipitation with tetraborate; however, the volumes of ethanol required for large-scale preparations are prohibitively large; therefore, method B is used for the latter. A preliminary trial indicates that method C may be a suitable compromise for small-scale analysis. After extraction of endocellular enzymes in a neutral buffer, the debri of unbroken cells and cell walls is collected by centrifugation at 10,000 g for 10 rain and extracted with 80% ethanol. KBr, unlike ethanol, interferes with spectrophotometric protein assay at 215/225 nm; therefore, the Warburg-Christian ~° method is used for method A. Large-Scale Purification Procedures. Kilogram quantities of dry milk, wheat germ, and dry yeast are used in large-scale purifications (Tables I III). Since most of the procedures are essentially the same for these materials, a general method is described. Step 1: Extraction. The dry material is suspended in 2-4 volumes of ethanol per weight in the stainless steel cup of a commercial Waring blendor and homogenized 2-4 min at full speed. The ensuing increase of homogenate temperature from about 20 to 40 ° is not detrimental to enzyme activity and may actually facilitate purification by denaturing unglycosylated proteins. The homogenate is slowly poured into a 30-cm-diameter bench-type B0chner funnel containing Whatman No. 1 paper while collecting the filtrate with the aid of an oil vacuum pump. The extraction is repeated, and filtrates are pooled. The filtrate may require clarification by additional filtration with Celite (diatomaceous earth). The Celite is washed on the filter with water and ethanol before use. Step 2: Tetraborate crystallization. Dry milk and wheat germ are extracted with 95% (w/v) ethanol and diluted to 80%. Na2B407 • 1OH20 and sodium acetate are slowly dissolved in the solution while stirring to 1 and 0.5% (w/v), respectively. Eighty percent ethanol is more efficient than 1o O. W a r b u r g and W. Christian, Biochem. Z. 310, 384 (1942).
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TABLE I PURIFICATION OF BOVINE MILK SOD S O D Activity Protein (mg)
Step 1. 2. 3. 4. 5. 6.
Ethanol extraction a Borate Recrystallization ( l x ) Recrystallization ( 4 x ) Dialysis Crystallization c
Units × 10 -6 U n i t s / m g
1,020 102 -92 2.8
6.1 6.5 4,350 5,600 12,000
6.0 6.4 2 6 4.3
× x × × ×
Purification factor
Recovery (-fold)
1b 10.7 3.3 × 103 9.9 x l03 7 x l05
1.1 713 918 2,000
103 104 107 107 l09
a F o u r liters o f 95% ethanol extract recovered from extraction of 1.8 kg dry milk. b 390-fold with r e s p e c t to total milk protein. c After dialyses 3 times at 20 °, the solution containing about 0.1 mg protein/ml was stored at 5 ° . T A B L E II PURIFICATION OF WHEAT GERM EXOCELLULAR SOD SOD Activity Step 1. 2. 3. 4. 5. 6.
Ethanol extract a Borate Ethanol Borate Dialysis Centrifugation
Volume (ml) 2,500 350 1,750 85 100 10
Protein (mg) 118,000 -470 138 2.6 0.4
Units × 10 -7
Units/mg
Purification factor
3.1 -10.9 3.2 5.0 3.5
2.6 × 102
1b
2.3 2.3 1.9 8.8
x x × x
105 104 107 107
8.8 8.8 7.3 3.4
× x x x
102 102 104 105
Recovery (-fold)
3.5 1.0 1.6 1.1
a F r o m 1 kg. b A b o u t 10-fold with respect to weight of wheat germ. T A B L E III PURIFICATION OF BAKER'S YEAST EXOCELLULAR SOD SOD Activity Step 1. 2. 3. 4. 5.
Ethanol extract a Borate Ethanol Borate Dialysis
Volume (ml)
Protein (mg)
1,600
2,500 --3.56 0.15
60 80
Units × 10 -6 U n i t s / m g 1.6 --11.7 3.4
F r o m 500 g dry yeast. b Purification was 213-fold with respect to cell dry weight. a
Purification factor
6.4 × 102
1b
3.3 × 106 2.3 x 107
5.2 × 103 3.6 x 104
Recovery (-fold)
7.3 2.1
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95% for yeast SOD extraction. Solid salts are slowly dissolved in the extract as described above. The tetraborate step is preferably carried out with a glass vessel because the crystals subsequently formed tenaciously stick to glass, a fact that facilitates their collection from the mother liquor. The ethanol-borate solution is allowed to sit overnight at 10°. The mother liquor is decanted and stored another day for the collection of a smaller crop of crystals. The crystals are collected in a minimum volume of 5 mM acetate buffer (pH 5.5) at room temperature. A glass rod or spatula is used to dislodge them from the glass vessel. Dissolution of the crystals is facilitated by magnetically stirring the solution on a hot plate while raising the temperature to 39-40 ° . At this stage of the process, either one of two tactics provide additional purification: recrystallization from buffer (Tables I, VI) or repetition of the ethanol-borate steps (Tables II-IV), another mode of recrystallization. The former tactic is more economical in time, effort, and material. Step 3: Recrystallization from buffer. After dissolving the crystals in buffer at 39°, the rate of subsequent crystal growth and the size of crystals are primarily regulated by the rate of temperature reduction. Addition of a few seed crystals or scratching the wall of the glass container with a glass rod sometimes hastens crystal growth. Large crystals are grown by storage of the solution several hours or overnight while decreasing the temperature slowly in increments from 20 to 10 to 5°. Rapid formation of small, birefringent crystals occurs if the solution is immediately set in an ice bath. Sham experiments indicate that tetraborate alone crystallizes under these conditions. Comparative analysis of the dry weight and protein content of the crystals indicates that the majority of the crystalline mass is sodium tetraborate. Step 4: Dialysis. Regardless of the method of recrystallization, subsequent dialysis enhances specific activity with respect to protein 10- to 100fold and removes excess borate salt (Tables I-IV). We suspect that low molecular weight glycopeptides are removed by dialysis. The amount of borate associated with the enzyme after dialysis has not been determined. Although tetraborate can be measured with the silver assay, the reactivity of proteins with silver precludes assay of protein-bound tetraborate. The crystals are dissolved as described and dialyzed against 200-400 volumes of distilled water at 20-25 ° with continuous stirring for 1 day. One or two additional dialyses are performed. The second but not the first dialysate is free of measurable tetraborate. The dialyzed samples are usually free of particulate matter. Dilute protein solutions are opaque and faintly blue.
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Step 5a: Crystallization from water. The ethanol and water solubilities of the enzyme indicate that it is an amphiphilic glycoprotein. After exhaustive dialysis against water at 20° , dilute enzyme protein solutions are opaque, unlike dilute or concentrated ethanolic solutions. That observation indicates a relatively low solubility in water. Although it is not known if tetraborate remains bound to the enzyme and influences its solubility, the situation indicates that it may also crystallize in a form at least relatively free of tetraborate. Indeed, after overnight storage at 5° the dialyzed milk enzyme (Table I) and some dialyzed yeast enzyme preparations formed crystals that subsequently grew to several millimeters in length. The crystalline milk SOD remains active in water at 5° for 2 years. Step 5b: Centrifugation. The dialyzed wheat germ enzyme preparation was exceptionally opaque although it contained only 26/zg protein/ml (Table II). It was centrifuged at 4° for 30 min at 30,000 g. The precipitate was dissolved in 10 ml of 0.2 M acetate buffer (pH 5.5). The solution contained 41/xg protein/ml and was highly opaque and slightly blue. The step recovered 70% of the activity and enriched specific activity about 4-fold (Table II). The absorbancy ratios at 215/225,280/260, and 280/560 nm were 1.27, 0.69, and 4.5, respectively. The 215/225 nm protein assay indicated 0.27 mg/ml; the 280/260 assay indicated 0.81 mg/ml. The latter probably overestimates concentration because of the exceptionally low 280/260 ratio, a general feature of the enzyme from several sources (Table VII). Affinity Chromatography. Highly purified yeast or milk SOD is bound to a column of concanavalin A-agarose in 10 mM acetate buffer (pH 5.5) containing 0.1 mM CaC12 and eluted with the buffer containing 0. I M a-methylmannoside. The binding and elution are nearly perfectly efficient as indicated by both protein and enzyme assays. The column must be washed thoroughly to ensure that the sample does not become contaminated with dissociable concanavalin. Without calcium chloride, the enzyme is not retained. This procedure might prove useful for the removal of possible trace amounts of bound tetraborate after dialysis. Affinity chromatography is not used for purification with respect to protein because it may not be sufficiently specific: most if not all exocellular proteins are probably glycoproteins. Purification of Calf Serum SOD. Serum presents special problems because it is extraordinarily rich in nonglycosylated protein. Three rather than one or two ethanol-borate cycles are necessary to increase the specific activity to a level comparable to that of the highly purified enzymes from other sources (Table IV). The gigantic mass of protein formed on adjusting serum to 80% (w/v) ethanol occludes a substantial amount of the solution volume after centrifugation. This problem is largely circum-
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T A B L E IV PURIFICATION OF BOVINE CALF SERUM EXOCELLULAR SOD SOD Activity
Volume
Protein
Step
(ml)
(mg)
Serum 1. Ethanol extract
250
185,000 --320 86 -1.1 0.20
2. 3. 4. 5. 6. 7.
Borate Ethanol Borate Ethanol Borate Dialysis
150 30 50 50
Purification Units x 10 -4 Units/mg 6.2 --7.2 30 -27 300
factor
Recovery (-fold)
0.33
1
2.4 x 102 3.5 × l03
7.3 × 102 1.1 × 104
1.2 4.8
2.5 × 105 1.5 x 107
8.3 x 105 4.5 x 107
4.4 48
vented by a preliminary fractionation with 50% (w/v) ethanol. The subsequent 50-80% ethanol step also must be clarified by centrifugation. Purification of Bacterial Exocellular SOD. Tables V and VI summarize purifications of the enzymes from dried cells and culture filtrates of Escherichia coli and Bacillus subtilis. It is not clear whether E. coli has genuine cell-bound ethanol-soluble EC-SOD. The attempt to precipitate it with borate failed, the only failure encountered among the various materials examined. Conversely, B. subtilus appears to possess a genuine cellbound enzyme. The results of purification of the enzyme from culture filtrates are relatively unequivocal (Table VI). Comparative analysis indicates that B. subtilis is a richer source (Tables V, VI).
TABLE V PURIFICATION OF EXOCELLULAR SOD FROM LYOPHILIZED BACTERIA SOD Activity *
Step 1. Ethanol
extract 2. Borate
E. B. E. B.
Volume
Protein c
Cells
(ml)
(mg/g)
coli subtilis coli subtilis
7.3 a 17 b 5 l0
4.46 19 0.52 0.47
Units x 10-4 Units/rag 3.6 47 0.1 230
8.1 2.5 1.9 4.9
x x x x
103 104 103 106
a One gram extracted with 10 mi of 85% ethanol; 7.3 ml recovered. b One gram extracted with 20 ml of 95% ethanol; 17.3 ml recovered. c Corrected for loss of extract solvent occluded with cells. d Purification was 50-fold with respect to cell weight.
Purification factor
Recovery (%)
1 Ia 0.2 196
2.7 489
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TABLE VI PURIFICATION OF BACTERIAL EXOCELLULARSOD FROM CULTURE FILTRATES SOD Activity Step 1. Culture filtrate a 2. Ethanol 3. Borate 4. Recrystallization (IX) 5. Recrystallization (2X)
Cells a
Volume (ml)
Protein (mg)
E B
50 50
E B E B B
50 50 5 50 10
375 1,600 -1.5 -0.38 1.0 0.145
Units × 10-4 Units/mg
Purification factor
Recovery (-fold)
0.65 21.4 -4.38
17.3 134
1 1
2.9 x 104
1.7 × 103
6.7
3.1 36.1 38
8.2 × 104 3.6 x 105 2.6 x 106
4.8 x 103 2.7 × 103 2 × 104
4.8 1.7 1.8
a E. coil (E) and B. subtilis (B) were grown overnight at 36° in shake cultures containing 50 ml of
medium with 2% glucose. The absorbancies at 600 nm of the cultures were 0.465 and 0.720 for E and B, respectively. After removal of cells by centrifugation, the supernatant was clarified by Millipore filtration. The culture medium was Vogel's minimal (R. H. Davis and F. J. de Serres, this series, Vol. 17A, p. 79).
Concanavalin A Inhibition of Yeast ExoceUular SOD in Vivo and in Vitro. Inhibition titrations of either highly purified or in vivo yeast EC-SOD with concanavaiin A (Con A) were performed. 6 Calcium chloride (0.1 mM) is essential for inhibition. The range of Con A concentrations for in vivo inhibition is much less than that which causes the cells to flocculate.
Discussion Purification methods and some enzyme properties briefly discussed in the preceding section are discussed at greater length here. The purification procedures are based on the assumption that all EC-SODs are large, highly glycosylated proteins; the previously demonstrated prototype is the human enzyme. 2 The literature on highly glycosylated proteins indicates that their properties resemble that of polysaccharides and sugars. Solubility and resistance to denaturation in organic solvents is one feature of highly glycosylated proteins exhibited by EC-SODs. They are soluble and remain active in 80-95% ethanol. The yeast enzyme is also soluble and fully active after extraction in 80% phenol. 6 Furthermore, unlike ordinary protein, EC-SODs are soluble in aqueous saturated ammonium sulfate. 6
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Sugars or glycoproteins form diol-charge complexes with borate) 1,~2 EC-SODs are relatively insoluble in the presence of tetraborate. The enzyme-tetraborate complex apparently cocrystallizes with free tetraborate salt from 80% ethanol or acidic acetate buffer. Since human and animal tissue, 3 bovine milk, and yeast EC-SODs bind to the plant lectin concanavalin A and are eluted with o~-methylmannoside, at least a portion of their polysaccharide probably consists of mannose, for which Con A is primarily specific. The human EC-SOD polypeptide subunit contains a hydrophobic amino-terminal amino acid sequence to residue 95 and a hydrophilic carboxy-terminal sequence at residues 194-222. 4 Assuming phylogenetic sequence homology, those features and bound polysaccharide probably account for the amphiphilic nature of EC-SODs observed here. The efficient extraction of the enzyme from cells with the chaotropic agents KBr or ethanol indicates that the hydrophobic region of the protein may bind to cell wall or plasmalemma. The inhibition of yeast EC-SOD in vivo by Con A indicates that it is bound to the cell wall surface. Inhibition of the isolated yeast enzyme by Con A may indicate that the lectin induces a conformational change. Yeast and calf serum EC-SOD exhibit fluorescence excitation and emission spectra characteristic of tryptophan-containing proteins6; however, the relatively low 280/260 absorbancy ratios (Table VII) indicate a relatively low tryptophan content, a feature that probably accounts for the consistent discrepancies between the two spectrophotometric methods of measurement of protein concentration. The specific activity of bovine red blood cell (RBC) SOD (endocellular) in the assay is about 6,000 units/mg protein. Conversely, the specific activities of the exocellular enzymes from various sources approach or slightly exceed that of RBC SOD in relatively impure samples and are 104to 106-fold more active in highly purified preparations (Tables I-VI). Marklund 2 compared the specific activities of human EC-SOD and the endocellular Cu,Zn-SOD and concluded that they are not appreciably different; however, the assay was at pH 7 and our results are at 10.8. The kinetic features are further clouded by our observations that the enzyme is frequently activated during the course of purification. Marklund 2 did not observe activation of the human enzyme during purification. The activation was sufficiently frequent and of sufficient magnitude (Tables I, III, IV, V) to indicate that it is not a spurious error. Removal of a naturally occurring inhibitor may account for the activation n j. X. Khym, this series, Vol. 12A, p. 93. ~2 j. R. Cann, this series, Vol. 25, p. 157.
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T A B L E VII ABSORBANCE RATIOS OF HIGHLY PURIFIED EXOCELLULAR SODs A b s o r b a n c e ratio, wavelength E n z y m e source Milk Calf s e r u m Wheat germ Yeast
280/260 n m 0.78 0.80 0.60 0.67
280/560 n m 2.8 3.0 ~ 4.7 5.6"
a hmax 560 n m in visible light.
effect. Even if the activation effect is discounted, the data indicate that the procedure provides excellent recoveries of activity and that specific activities are enriched many orders of magnitude with respect to ethanolsoluble protein. Moreover, the initial extraction alone enriches specific activity 10- to 400-fold with respect to biomass (Tables I-III, V). Although activity recoveries are high, protein yields are low, ranging from 0.15 to 2.8 mg (Tables I-IV); hence, either larger scale preparations or microanalytical techniques will be required for chemical analyses. The ready tendency of the enzymes to crystallize from dilute solutions, with or without the addition of borate, indicates that crystallographic analysis of their structure may be feasible. The highly purified enzyme solutions are faintly blue and exhibit significant 280/560 absorbancy ratios (Table VII). The visible absorbancy spectra of the calf and yeast enzymes exhibit a peak at 560 nm. 6 The yeast and calf enzymes are inhibited by 1 mM cyanide. 6 Collectively, those observations indicate that the enzymes contain copper, as proved for the human enzyme. 2 An attempt by a commercial analytical laboratory to determine the isoelectric points and molecular weights of the calf and yeast enzyme subunits by the O'Farrell method was unsuccessful, apparently because the proteins failed to stain with Coomassie blue. 6 Specific glycoprotein stains may be required. The long-range research goals motivating this study, noted in the Introduction, are now approachable. Minute quantities of milk or yeast EC-SODs are active in therapy of physiological and biochemical abnormalities of EC-SOD-deficient mutants of Neurospora and yeast. 6 These findings have permitted development of quantitative bioassays of the enzymes and discovery of the probable biological function of the enzyme. 6
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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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In this view the difference between the green and the pink complexes is that the former contains oxidized Desferal while the latter contains intact Desferal. Oxidation of one of its hydroxamate ligand groups should decrease the affinity of the Desferal for Mn(III), and, as expected, the green complex was less stable to EDTA than was the pink complex. Desferal is known to act as an electron donor, and the nitroxide product of the univalent oxidation of one of its hydroxamate groups has been studied by electron paramagnetic resonance. ~° Since the pink complex was prepared by reacting 1.0 mol ascorbate per mole of Desferal with a 25% molar excess of MnO2, it must be presumed to contain dehydroascorbate. Dehydroascorbate at 250 /xM dd not inhibit the reduction of cytochrome c by the flux of 02- generated by the action of xanthine oxidase on 10 mM CH3CHO. It follows that this concentration of dehydroascorbate did not exhibit any detectable ability to intercept 02- and that the very much lower concentration (4/zM) present during assays of the pink complex would not interfere with assays of the activity of that complex. The greater stability and catalytic activity of the pink complex suggest that it may be a more useful mimic of SOD activity than the green complex. This remains to be explored in vivo. Acknowledgments This work was supported by research grants from the National Science Foundation, the Council for Tobacco Research--USA, Inc., and the National Institutes of Health. 10 K. M. Morehouse, W. D. Flitter, and R. P. Mason, FEBS Lett. 222, 246 (1987).
[24] Purification of E x o 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 s
By KENNETH D. MUNKRES Introduction
Although superoxide dismutases (SODs, EC 1.15.1.1) have been intensively investigated for many years, the existence of exocellular forms of the enzyme was recognized only in the 1980s. The existence of exocellular forms of the enzyme (EC-SODs) in bovine milk was discovered almost simultaneously in three laboratories around 1975. ~ Subsequently, 1 M. Korycka-Dahl, T. Richardson, and C. L. Hicks, J. Food Prot. 42, 867 (1979).
METHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990 by Acadenfic Press, Inc. All rights of reproduction in any form reserved.
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Marklund and associates 2-5 reported a series of investigations of EC-SODs from human and animal tissues. Marklund 2 purified human lung EC-SOD on a large scale with low yield by a lengthy series of ion-exchange and affinity chromatographic steps. Subsequently, the human EC-SOD structural gene was cloned, and the enzyme was synthesized in large quantities by genetic engineering techniques. 4,5 The complete amino acid sequence of the polypeptide subunit and signal peptide of the enzyme was deduced from the nucleotide sequence of the gene. Comparison of sequence similarities indicated that the exo- and endocellular SOD isozymes arose from a common ancestral enzyme before the evolution of fungi and plants; hence, one may infer a broad phylogenetic distribution of the exoenzyme. The results of this investigation support that inference. Facile procedures for large-scale purification and crystallization were invented and applied to bovine milk and blood serum, wheat germ, yeast, and bacteria. The enzyme was also partially purified from Neurospora. 6 The widespread phylogenetic distribution of the enzyme indicates that it plays an essential but yet unknown biological role. Knowledge of its biological role may be significant for application of the engineered human enzyme in proposed clinical therapy of human diseases mediated by free radicals. Our laboratory is investigating the genetic regulation and biological function of EC-SOD. EC-SOD-deficient mutants have been isolated in N e u r o s p o r a and yeast. 6 The aim of this study was to develop the technology to conduct comparative EC-SOD therapy of the physiological "diseases" of the mutants. Human EC-SOD is a tetrameric glycoprotein whose molecular weight is 135,000. 2 The present purification procedures are designed to select proteins of this nature. Materials Defatted, pasteurized dry milk (Carnation Co., Los Angeles, CA), natural (untoasted) wheat germ (Hodgson Mill Enterprise, Gainsville, MO), and Red Star Quick-Rise dried bakers' yeast in vacuo (Universal Foods Corp., Milwaukee, WI) were obtained from local grocers. Lyophi-
2 S. L. Marklund, Proc. Natl. Acad. Sci. U.S.A. 79, 7634 (1982). 3 S. L. Marklund, Biochem. J. (London) 222, 649 (1984). 4 K. Hjalmarsson, S. L. Marklund, A,. Engstr6m, and T. Edlund, Proc. Natl. Acad. Sci. U.S.A. 84, 6340 (1987). 5 L. Tibell, K. Hjalmarsson, T. Edlund, G. Skogman, A. Engstr6m, and S. L. Marklund, Proc. Natl. Acad. Sci. U.S.A. 84, 6634 (1987). K. D. Munkres, unpublished (1988).
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lized cells of Bacillus subtilis (ATCC 6633) and Escherichia coli (strain W) and concanavalin A-agarose were obtained from Sigma Chemical Co. (St. Louis, MO). Sterile calf serum was from HyClone Laboratories (Logan, UT). Methods and Results
SOD Assay. The spectrophotometric method of Misra and Fridovich7 is used. The method is based on the inhibition by the enzyme of the spontaneous, alkali-catalyzed autoxidation of epinephrine to norepinephrine. Enzyme in 10-100/zl of distilled water diluent is added to 1 ml of the reaction mixture in a semimicrocuvette at 36°. The reaction rate is monitored in a Gilford recording spectrophotometer at 480 nm for 6 min. After a lag period of 1 to 3 min, the linear rate during the next 2 to 3 min is graphically determined. Plots of percent inhibition versus protein concentration are constructed with two or three points, and the concentration producing 50% inhibition (1 unit) is determined by interpolation. Usually the plots are linear from 20 to 80% inhibition. Rarely some samples exhibit linear plots that plateau at 40-60% inhibition. In those instances, a unit of activity is defined as that concentration producing half-maximal inhibition. Employing commercial bovine red blood cell SOD, we confirmed the observation of Misra 7,8 that the present assay method is about 2-fold more sensitive than the xanthine oxidase method of McCord and Fridovich. Ethanol mimics SOD: at least 1% inhibits the assay. Ethanol extracts should contain sufficient enzyme to permit its assay after dilution in water to noninhibitory ethanol concentrations. Tetraborate does not interfere with the assay. Protein Assay. The spectrophotometric method of Murphy and Kies 9 is used. Tetraborate Assay. The course of dialysis to remove tetraborate is monitored by a colorimetric assay. A sample of the dialysate (0.9 ml) is mixed with 0.1 ml of 1% silver nitrate and incubated at room temperature about 10 min or until the absorbancy at 500 nm becomes constant. The extinction coefficient of tetraborate (Emu, cm -j) is 0.255. The assay detects as little as 10-100/zg sodium tetraborate per milliliter. Extraction of Exocellular SOD. Survey of 16 strains of bakers', beer, and wine yeast indicated that Red Star Quick-Rise Bakers' yeast is an 7 H. P. Misra and I. Fridovich, J. Biol. Chem. 247, 3410 (1972). 8 H. P. Misra, in "CRC Handbook of Methods for Oxygen Radical Research" (R. A. Greenwald, ed.), p. 237. CRC Press, Boca Raton, Florida, 1985. 9 j. B. Murphy and M. W. Kies, Biochim. Biophys. Acta 45, 382 (1960).
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extraordinarily rich source of EC-SOD. 6 The EC-SOD activity of the yeast in vioo is determined by assay with washed cells diluted in water. The specific activity is about 0.89 units/mg dry cells. Two methods efficiently extract the enzyme, as indicated by either units extracted or units remaining with the cell. Method A uses 0.3 M KBr in 5 mM acetate buffer (pH 5.5); method B uses 80% ethanol. One gram of dry yeast is stirred 10-20 min with 10-20 volumes of extraction solvent at room temperature. Cells are removed by centrifugation at I0,000 g for 10 min. Two extractions yield at least 95% of the enzyme units initially present. Method A is preferred if we wish to subsequently break the cells and measure endocellular SOD activities. The exoenzyme may be concentrated and purified from the extract on a small scale by adjusting the extract to 80% ethanol followed by precipitation with tetraborate; however, the volumes of ethanol required for large-scale preparations are prohibitively large; therefore, method B is used for the latter. A preliminary trial indicates that method C may be a suitable compromise for small-scale analysis. After extraction of endocellular enzymes in a neutral buffer, the debri of unbroken cells and cell walls is collected by centrifugation at 10,000 g for 10 rain and extracted with 80% ethanol. KBr, unlike ethanol, interferes with spectrophotometric protein assay at 215/225 nm; therefore, the Warburg-Christian ~° method is used for method A. Large-Scale Purification Procedures. Kilogram quantities of dry milk, wheat germ, and dry yeast are used in large-scale purifications (Tables I III). Since most of the procedures are essentially the same for these materials, a general method is described. Step 1: Extraction. The dry material is suspended in 2-4 volumes of ethanol per weight in the stainless steel cup of a commercial Waring blendor and homogenized 2-4 min at full speed. The ensuing increase of homogenate temperature from about 20 to 40 ° is not detrimental to enzyme activity and may actually facilitate purification by denaturing unglycosylated proteins. The homogenate is slowly poured into a 30-cm-diameter bench-type B0chner funnel containing Whatman No. 1 paper while collecting the filtrate with the aid of an oil vacuum pump. The extraction is repeated, and filtrates are pooled. The filtrate may require clarification by additional filtration with Celite (diatomaceous earth). The Celite is washed on the filter with water and ethanol before use. Step 2: Tetraborate crystallization. Dry milk and wheat germ are extracted with 95% (w/v) ethanol and diluted to 80%. Na2B407 • 1OH20 and sodium acetate are slowly dissolved in the solution while stirring to 1 and 0.5% (w/v), respectively. Eighty percent ethanol is more efficient than 1o O. W a r b u r g and W. Christian, Biochem. Z. 310, 384 (1942).
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TABLE I PURIFICATION OF BOVINE MILK SOD S O D Activity Protein (mg)
Step 1. 2. 3. 4. 5. 6.
Ethanol extraction a Borate Recrystallization ( l x ) Recrystallization ( 4 x ) Dialysis Crystallization c
Units × 10 -6 U n i t s / m g
1,020 102 -92 2.8
6.1 6.5 4,350 5,600 12,000
6.0 6.4 2 6 4.3
× x × × ×
Purification factor
Recovery (-fold)
1b 10.7 3.3 × 103 9.9 x l03 7 x l05
1.1 713 918 2,000
103 104 107 107 l09
a F o u r liters o f 95% ethanol extract recovered from extraction of 1.8 kg dry milk. b 390-fold with r e s p e c t to total milk protein. c After dialyses 3 times at 20 °, the solution containing about 0.1 mg protein/ml was stored at 5 ° . T A B L E II PURIFICATION OF WHEAT GERM EXOCELLULAR SOD SOD Activity Step 1. 2. 3. 4. 5. 6.
Ethanol extract a Borate Ethanol Borate Dialysis Centrifugation
Volume (ml) 2,500 350 1,750 85 100 10
Protein (mg) 118,000 -470 138 2.6 0.4
Units × 10 -7
Units/mg
Purification factor
3.1 -10.9 3.2 5.0 3.5
2.6 × 102
1b
2.3 2.3 1.9 8.8
x x × x
105 104 107 107
8.8 8.8 7.3 3.4
× x x x
102 102 104 105
Recovery (-fold)
3.5 1.0 1.6 1.1
a F r o m 1 kg. b A b o u t 10-fold with respect to weight of wheat germ. T A B L E III PURIFICATION OF BAKER'S YEAST EXOCELLULAR SOD SOD Activity Step 1. 2. 3. 4. 5.
Ethanol extract a Borate Ethanol Borate Dialysis
Volume (ml)
Protein (mg)
1,600
2,500 --3.56 0.15
60 80
Units × 10 -6 U n i t s / m g 1.6 --11.7 3.4
F r o m 500 g dry yeast. b Purification was 213-fold with respect to cell dry weight. a
Purification factor
6.4 × 102
1b
3.3 × 106 2.3 x 107
5.2 × 103 3.6 x 104
Recovery (-fold)
7.3 2.1
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95% for yeast SOD extraction. Solid salts are slowly dissolved in the extract as described above. The tetraborate step is preferably carried out with a glass vessel because the crystals subsequently formed tenaciously stick to glass, a fact that facilitates their collection from the mother liquor. The ethanol-borate solution is allowed to sit overnight at 10°. The mother liquor is decanted and stored another day for the collection of a smaller crop of crystals. The crystals are collected in a minimum volume of 5 mM acetate buffer (pH 5.5) at room temperature. A glass rod or spatula is used to dislodge them from the glass vessel. Dissolution of the crystals is facilitated by magnetically stirring the solution on a hot plate while raising the temperature to 39-40 ° . At this stage of the process, either one of two tactics provide additional purification: recrystallization from buffer (Tables I, VI) or repetition of the ethanol-borate steps (Tables II-IV), another mode of recrystallization. The former tactic is more economical in time, effort, and material. Step 3: Recrystallization from buffer. After dissolving the crystals in buffer at 39°, the rate of subsequent crystal growth and the size of crystals are primarily regulated by the rate of temperature reduction. Addition of a few seed crystals or scratching the wall of the glass container with a glass rod sometimes hastens crystal growth. Large crystals are grown by storage of the solution several hours or overnight while decreasing the temperature slowly in increments from 20 to 10 to 5°. Rapid formation of small, birefringent crystals occurs if the solution is immediately set in an ice bath. Sham experiments indicate that tetraborate alone crystallizes under these conditions. Comparative analysis of the dry weight and protein content of the crystals indicates that the majority of the crystalline mass is sodium tetraborate. Step 4: Dialysis. Regardless of the method of recrystallization, subsequent dialysis enhances specific activity with respect to protein 10- to 100fold and removes excess borate salt (Tables I-IV). We suspect that low molecular weight glycopeptides are removed by dialysis. The amount of borate associated with the enzyme after dialysis has not been determined. Although tetraborate can be measured with the silver assay, the reactivity of proteins with silver precludes assay of protein-bound tetraborate. The crystals are dissolved as described and dialyzed against 200-400 volumes of distilled water at 20-25 ° with continuous stirring for 1 day. One or two additional dialyses are performed. The second but not the first dialysate is free of measurable tetraborate. The dialyzed samples are usually free of particulate matter. Dilute protein solutions are opaque and faintly blue.
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Step 5a: Crystallization from water. The ethanol and water solubilities of the enzyme indicate that it is an amphiphilic glycoprotein. After exhaustive dialysis against water at 20° , dilute enzyme protein solutions are opaque, unlike dilute or concentrated ethanolic solutions. That observation indicates a relatively low solubility in water. Although it is not known if tetraborate remains bound to the enzyme and influences its solubility, the situation indicates that it may also crystallize in a form at least relatively free of tetraborate. Indeed, after overnight storage at 5° the dialyzed milk enzyme (Table I) and some dialyzed yeast enzyme preparations formed crystals that subsequently grew to several millimeters in length. The crystalline milk SOD remains active in water at 5° for 2 years. Step 5b: Centrifugation. The dialyzed wheat germ enzyme preparation was exceptionally opaque although it contained only 26/zg protein/ml (Table II). It was centrifuged at 4° for 30 min at 30,000 g. The precipitate was dissolved in 10 ml of 0.2 M acetate buffer (pH 5.5). The solution contained 41/xg protein/ml and was highly opaque and slightly blue. The step recovered 70% of the activity and enriched specific activity about 4-fold (Table II). The absorbancy ratios at 215/225,280/260, and 280/560 nm were 1.27, 0.69, and 4.5, respectively. The 215/225 nm protein assay indicated 0.27 mg/ml; the 280/260 assay indicated 0.81 mg/ml. The latter probably overestimates concentration because of the exceptionally low 280/260 ratio, a general feature of the enzyme from several sources (Table VII). Affinity Chromatography. Highly purified yeast or milk SOD is bound to a column of concanavalin A-agarose in 10 mM acetate buffer (pH 5.5) containing 0.1 mM CaC12 and eluted with the buffer containing 0. I M a-methylmannoside. The binding and elution are nearly perfectly efficient as indicated by both protein and enzyme assays. The column must be washed thoroughly to ensure that the sample does not become contaminated with dissociable concanavalin. Without calcium chloride, the enzyme is not retained. This procedure might prove useful for the removal of possible trace amounts of bound tetraborate after dialysis. Affinity chromatography is not used for purification with respect to protein because it may not be sufficiently specific: most if not all exocellular proteins are probably glycoproteins. Purification of Calf Serum SOD. Serum presents special problems because it is extraordinarily rich in nonglycosylated protein. Three rather than one or two ethanol-borate cycles are necessary to increase the specific activity to a level comparable to that of the highly purified enzymes from other sources (Table IV). The gigantic mass of protein formed on adjusting serum to 80% (w/v) ethanol occludes a substantial amount of the solution volume after centrifugation. This problem is largely circum-
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T A B L E IV PURIFICATION OF BOVINE CALF SERUM EXOCELLULAR SOD SOD Activity
Volume
Protein
Step
(ml)
(mg)
Serum 1. Ethanol extract
250
185,000 --320 86 -1.1 0.20
2. 3. 4. 5. 6. 7.
Borate Ethanol Borate Ethanol Borate Dialysis
150 30 50 50
Purification Units x 10 -4 Units/mg 6.2 --7.2 30 -27 300
factor
Recovery (-fold)
0.33
1
2.4 x 102 3.5 × l03
7.3 × 102 1.1 × 104
1.2 4.8
2.5 × 105 1.5 x 107
8.3 x 105 4.5 x 107
4.4 48
vented by a preliminary fractionation with 50% (w/v) ethanol. The subsequent 50-80% ethanol step also must be clarified by centrifugation. Purification of Bacterial Exocellular SOD. Tables V and VI summarize purifications of the enzymes from dried cells and culture filtrates of Escherichia coli and Bacillus subtilis. It is not clear whether E. coli has genuine cell-bound ethanol-soluble EC-SOD. The attempt to precipitate it with borate failed, the only failure encountered among the various materials examined. Conversely, B. subtilus appears to possess a genuine cellbound enzyme. The results of purification of the enzyme from culture filtrates are relatively unequivocal (Table VI). Comparative analysis indicates that B. subtilis is a richer source (Tables V, VI).
TABLE V PURIFICATION OF EXOCELLULAR SOD FROM LYOPHILIZED BACTERIA SOD Activity *
Step 1. Ethanol
extract 2. Borate
E. B. E. B.
Volume
Protein c
Cells
(ml)
(mg/g)
coli subtilis coli subtilis
7.3 a 17 b 5 l0
4.46 19 0.52 0.47
Units x 10-4 Units/rag 3.6 47 0.1 230
8.1 2.5 1.9 4.9
x x x x
103 104 103 106
a One gram extracted with 10 mi of 85% ethanol; 7.3 ml recovered. b One gram extracted with 20 ml of 95% ethanol; 17.3 ml recovered. c Corrected for loss of extract solvent occluded with cells. d Purification was 50-fold with respect to cell weight.
Purification factor
Recovery (%)
1 Ia 0.2 196
2.7 489
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TABLE VI PURIFICATION OF BACTERIAL EXOCELLULARSOD FROM CULTURE FILTRATES SOD Activity Step 1. Culture filtrate a 2. Ethanol 3. Borate 4. Recrystallization (IX) 5. Recrystallization (2X)
Cells a
Volume (ml)
Protein (mg)
E B
50 50
E B E B B
50 50 5 50 10
375 1,600 -1.5 -0.38 1.0 0.145
Units × 10-4 Units/mg
Purification factor
Recovery (-fold)
0.65 21.4 -4.38
17.3 134
1 1
2.9 x 104
1.7 × 103
6.7
3.1 36.1 38
8.2 × 104 3.6 x 105 2.6 x 106
4.8 x 103 2.7 × 103 2 × 104
4.8 1.7 1.8
a E. coil (E) and B. subtilis (B) were grown overnight at 36° in shake cultures containing 50 ml of
medium with 2% glucose. The absorbancies at 600 nm of the cultures were 0.465 and 0.720 for E and B, respectively. After removal of cells by centrifugation, the supernatant was clarified by Millipore filtration. The culture medium was Vogel's minimal (R. H. Davis and F. J. de Serres, this series, Vol. 17A, p. 79).
Concanavalin A Inhibition of Yeast ExoceUular SOD in Vivo and in Vitro. Inhibition titrations of either highly purified or in vivo yeast EC-SOD with concanavaiin A (Con A) were performed. 6 Calcium chloride (0.1 mM) is essential for inhibition. The range of Con A concentrations for in vivo inhibition is much less than that which causes the cells to flocculate.
Discussion Purification methods and some enzyme properties briefly discussed in the preceding section are discussed at greater length here. The purification procedures are based on the assumption that all EC-SODs are large, highly glycosylated proteins; the previously demonstrated prototype is the human enzyme. 2 The literature on highly glycosylated proteins indicates that their properties resemble that of polysaccharides and sugars. Solubility and resistance to denaturation in organic solvents is one feature of highly glycosylated proteins exhibited by EC-SODs. They are soluble and remain active in 80-95% ethanol. The yeast enzyme is also soluble and fully active after extraction in 80% phenol. 6 Furthermore, unlike ordinary protein, EC-SODs are soluble in aqueous saturated ammonium sulfate. 6
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Sugars or glycoproteins form diol-charge complexes with borate) 1,~2 EC-SODs are relatively insoluble in the presence of tetraborate. The enzyme-tetraborate complex apparently cocrystallizes with free tetraborate salt from 80% ethanol or acidic acetate buffer. Since human and animal tissue, 3 bovine milk, and yeast EC-SODs bind to the plant lectin concanavalin A and are eluted with o~-methylmannoside, at least a portion of their polysaccharide probably consists of mannose, for which Con A is primarily specific. The human EC-SOD polypeptide subunit contains a hydrophobic amino-terminal amino acid sequence to residue 95 and a hydrophilic carboxy-terminal sequence at residues 194-222. 4 Assuming phylogenetic sequence homology, those features and bound polysaccharide probably account for the amphiphilic nature of EC-SODs observed here. The efficient extraction of the enzyme from cells with the chaotropic agents KBr or ethanol indicates that the hydrophobic region of the protein may bind to cell wall or plasmalemma. The inhibition of yeast EC-SOD in vivo by Con A indicates that it is bound to the cell wall surface. Inhibition of the isolated yeast enzyme by Con A may indicate that the lectin induces a conformational change. Yeast and calf serum EC-SOD exhibit fluorescence excitation and emission spectra characteristic of tryptophan-containing proteins6; however, the relatively low 280/260 absorbancy ratios (Table VII) indicate a relatively low tryptophan content, a feature that probably accounts for the consistent discrepancies between the two spectrophotometric methods of measurement of protein concentration. The specific activity of bovine red blood cell (RBC) SOD (endocellular) in the assay is about 6,000 units/mg protein. Conversely, the specific activities of the exocellular enzymes from various sources approach or slightly exceed that of RBC SOD in relatively impure samples and are 104to 106-fold more active in highly purified preparations (Tables I-VI). Marklund 2 compared the specific activities of human EC-SOD and the endocellular Cu,Zn-SOD and concluded that they are not appreciably different; however, the assay was at pH 7 and our results are at 10.8. The kinetic features are further clouded by our observations that the enzyme is frequently activated during the course of purification. Marklund 2 did not observe activation of the human enzyme during purification. The activation was sufficiently frequent and of sufficient magnitude (Tables I, III, IV, V) to indicate that it is not a spurious error. Removal of a naturally occurring inhibitor may account for the activation n j. X. Khym, this series, Vol. 12A, p. 93. ~2 j. R. Cann, this series, Vol. 25, p. 157.
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T A B L E VII ABSORBANCE RATIOS OF HIGHLY PURIFIED EXOCELLULAR SODs A b s o r b a n c e ratio, wavelength E n z y m e source Milk Calf s e r u m Wheat germ Yeast
280/260 n m 0.78 0.80 0.60 0.67
280/560 n m 2.8 3.0 ~ 4.7 5.6"
a hmax 560 n m in visible light.
effect. Even if the activation effect is discounted, the data indicate that the procedure provides excellent recoveries of activity and that specific activities are enriched many orders of magnitude with respect to ethanolsoluble protein. Moreover, the initial extraction alone enriches specific activity 10- to 400-fold with respect to biomass (Tables I-III, V). Although activity recoveries are high, protein yields are low, ranging from 0.15 to 2.8 mg (Tables I-IV); hence, either larger scale preparations or microanalytical techniques will be required for chemical analyses. The ready tendency of the enzymes to crystallize from dilute solutions, with or without the addition of borate, indicates that crystallographic analysis of their structure may be feasible. The highly purified enzyme solutions are faintly blue and exhibit significant 280/560 absorbancy ratios (Table VII). The visible absorbancy spectra of the calf and yeast enzymes exhibit a peak at 560 nm. 6 The yeast and calf enzymes are inhibited by 1 mM cyanide. 6 Collectively, those observations indicate that the enzymes contain copper, as proved for the human enzyme. 2 An attempt by a commercial analytical laboratory to determine the isoelectric points and molecular weights of the calf and yeast enzyme subunits by the O'Farrell method was unsuccessful, apparently because the proteins failed to stain with Coomassie blue. 6 Specific glycoprotein stains may be required. The long-range research goals motivating this study, noted in the Introduction, are now approachable. Minute quantities of milk or yeast EC-SODs are active in therapy of physiological and biochemical abnormalities of EC-SOD-deficient mutants of Neurospora and yeast. 6 These findings have permitted development of quantitative bioassays of the enzymes and discovery of the probable biological function of the enzyme. 6
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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
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