ASSAYS IN W H O L E B A C T E R I A L C E L L S
It is possible to prepare 40 to 60 erythrocyte samples for analysis per day. The automated method allows for the analysis of 25 samples in 6 min in contrast to time-consuming manual methods that require large sample volumes. 3 In the present method, the pH for the assay was increased to 10 from 7.8, 4 thus increasing the sensitivity of the assay for Cu,Zn-SOD 17 times. The reduction of cytochrome c was followed at the more sensitive 415 nm compared to 550 nm used in most assays. 1,3 With these modifications, the sensitivity of the assay was increased 28-fold. Using this method, a standard preparation of bovine liver SOD was found to have an activity of 90,560 U/mg of protein compared to the reported activity of 3200 U/mg. Thus, the units of activity calculated with the present assay are equivalent to 0.035 U reported by Sigma. This does not affect the use of bovine liver SOD as standards since they are used only to calculate the slope. A unit of SOD activity is arbitrarily defined as that activity which will decrease the rate of cytochrome c reduction by 50% under standard assay conditions. 3 L. Floh6 and F. 0tting, this series, Voi. 105, p. 93.
4 C. O. Beauchamp and I. Fridovich, Biochim. Biophys. Acta 317, 50 (1973).
 A s s a y o f S u p e r o x i d e D i s m u t a s e A p p l i c a b l e to W h o l e B a c t e r i a l Cells
By F. S. ARCHIBALD Since 1968 a wide variety of assays for the quantitation of superoxide dismutase (SOD) activity have been proposed and employed. Most are indirect assays requiring both a generator of a known flux of 02- and a chemical detector of the flux. SOD is then quantitated indirectly in a sample by the ability of that sample to reduce the detected O2- flux. Such assays have proved suitable for purified SOD and for most crude, dialyzed broken cell preparations. The present alternatives to this sort of indirect SOD assay, for example, direct viewing of O2- in the far-UV, electron paramagnetic resonance using free radical spin traps such as DMPO (5,5'-dimethyl-l-pyrroline 1-oxide), 19F NMR, stopped-flow spectroscopy, and chemiluminescence, are not practical with the usual broken cell extracts employed and SOD levels found in biological systems. Given the need for a controllable, known flux of 02- and known amounts of an O2- detector in all present assays for SOD in crude cell extracts, it is probably not possible to develop an "intact cell" bacterial METHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990 by Academic Press, Inc. All fights of reproduction in any form reserved.
ASSAY OF FORMATION OR REMOVAL OF OXYGEN RADICALS
SOD assay because (1) the cytoplasmic membrane (phospholipid bilayer) is impermeable to Oz- and most macromolecules and (2) SOD is always found within the cytoplasmic membrane. Thus, a usable whole cell assay must permeabilize the cells, allowing entry of Oz- and the Oz- detector or egress of SOD. Unfortunately, many bacteria, especially gram-positive rods and cocci have heavy walls, and are broken quantitatively only by harsh, laborintensive methods such as the hydraulic (French) press alone or combined with freeze-thaw or ball and glass bead mills. This creates severe problems in quantitating SOD in genetic and other studies that produce large numbers of small samples. It also prevents the screening of large numbers of bacterial colonies or clones on solid media for individuals unusually high or low in SOD. Lacking heavy walls, most animal cells readily lyse under mild physical, osmotic, or chemical insult, reducing the problem, but for many plant and protistan cell types (e.g., algae and fungi) the problem of accessibility also exists.
Assay of Toluenized Whole Bacterial Cells for Superoxide Dismutase Treatment of whole prokaryotic cells with toluene results in loss of membrane integrity and exit of cytoplasmic proteins and small molecules, although the cells often retain a normal microscopic appearance. Toluene permeabilization has been used for the quantitation of specific cytoplasmic proteins in a number of prokaryotes and yeasts.l-5 The following is a modification of the light- and riboflavin-driven hydroxylamine-nitrite SOD assay of Whitelam and Codd 5 as used on the toluenized cells of several bacterial species. Method. Cells are harvested from agar plate or broth cultures, washed with an appropriate buffer (e.g., 25 mM Tris base, I mM sodium EDTA, pH 8.0, for neisserial species), and resuspended to a cell protein concentration of 0.1-1.0 mg/ml. To 4 ml of the cell suspension is added 2 ml of toluene. The suspension shaken for 5 min, placed on ice, and the aqueous phase recovered. A 0.2 ml sample of the aqueous toluenized cell phase is added to a tube containing 5 ml of the Tris buffer, 10/.~M flavin mononucleotide (FMN), and 5 mM hydroxylamine. The tubes are incubated (25°) under uniform cool white fluorescent light (-80 foot-candles or 860 lux) i R. 2 F. 3 E. 4 F. 5 G.
Serrano, H. M. Gancedo, and D. Gancedo, Eur. J. Biochem. 34, 479 (1973). R. Tabita, P. Caruso, and W. Whitman, Anal. Biochem. 84, 462 (1978). Flores, M. G. Guerrero, and M. Losada, Arch. Microbiol. 128, 137 (1980). S. Archibald and M.-N. Duong, Infect. lmmun. 51, 631 (1986). C. Whitelam and G. A. Codd, Anal. Biochem. 121, 207 (1982).
S O D ASSAYS IN WHOLE BACTERIAL CELLS
for 25 min on a white background. The incubation area should be checked with a photometer for uniformity of illumination. A 1.0 ml sample of each assay mixture is assayed for nitrite formation by the addition of 1.0 ml of 20 mM sulfanilic acid and 1.0 ml of 7 mM anaphthylamine, both in 4.2 M acetic acid. These reagents are made by dissolving 0.5 g a-naphthylamine in 100 ml boiling water, to which, after cooling to 25 °, is added 125 ml acetic acid, and the volume is made up to 500 ml with cold water; the reagent is stored in the dark. The sulfanilic acid (1.7 g) is dissolved in 375 ml of warm water, and 125 ml of acetic acid is added. The assay mixture is incubated for 20 min, and the colored diazo complex is quantitated at 530 nm on a good spectrophotometer. Initial controls should include several concentrations of purified SOD protein, boiled cells, buffer blanks, and a SOD assay on mechanically broken cells to check the efficiency of SOD release by toluene from the cell type being assayed. Principle. The assay depends on the production of 02- by light, FMN, and EDTA and by the 02--mediated oxidation of hydroxylamine to nitrite by 02-. The oxidation of hydroxylamine occurs in two steps, both of which are sensitive to inhibition by SOD, NH2OH + 02- + H + ---> "NHOH + H202 • NHOH + 02- ~ NO2- + H20
The nitrite produced is quantified in a conventional assay in which NOzreacts with sulfanilic acid in an acid solution to form a diazo compound. This compound is then reacted with a-naphthylamine to form a red complex readily measured at 530 nm. Care should be exercised in the handling of a-naphthylamine as it has been identified as a carcinogen. The system will detect NO2- levels ranging from less than 10 to 180 ng/ml. C a v e a t s . Although a number of workers have reported the hydroxylamine-NO2- reaction to be a useful detector in crude extracts, 4-s Bielski et al. 9 have presented evidence from pulse radiolysis studies that the initial reaction of 02- and hydroxylamine is in fact much slower (in a defined buffer-formate-water system) than it appears to be in actual samples. They speculate that this discrepancy is due to modulation of the initial reaction rate by metals, namely, the observed sample inhibition of NO2- formation may be dependent on a factor other than the SOD content of the sample. However, comparison of the results with those obtained using other SOD assay methods, demonstration of SOD-inhibitable 6 E. F. Elstner and A. Heupel, Anal. Biochem. 70, 616 (1977). 7 y . Kono, Arch. Biochem. Biophys. 186, 189 (1978). s W. Bors, C. Michel, and M. Saran, Z. Naturforsch., C: Biosci. 33, 891 (1978). 9 B. H. J. Bielski, R. L. Arudi, D. E, Cabelli, and W. Bors, Anal. Biochem. 142, 207 (1984).
ASSAY OF FORMATION OR REMOVAL OF OXYGEN RADICALS
and O2--dependent hydroxylamine oxidation in metal-poor Tris-buffered controls, and the absence of hydroxylamine oxidation inhibition in boiled cell assay controls 4-8 all argue for the observed NO2- production being dependent on 02- and largely or entirely inhibitable by SOD. Alternative Assays
The NADH, PMS (phenazine methosulfonate), and NBT (nitro blue tetrazolium) 02- generator and detector system described by Nishikimi et al. 1° can reportedly be substituted for the light-driven F M N - E D T A hydroxylamine system for toluenized cells described above with satisfactory results.5 In this system NADH reduces PMS which autoxidizes in the presence of Oz to yield 02-. This NADH-PMS-driven reduction of NBT to its blue insoluble monoformazan is 95% inhibitable by SOD, suggesting that 5% of the NBT reduction is due to direct reaction with reduced PMS. In contrast, cytochrome e added to reduced PMS is almost entirely reduced by an Oz--independent mechanism. As with other SOD assays employing nitro blue tetrazolium (NBT) as the O2- detector, the analyst should keep in mind that this tetrazolium dye can be oxidized and reduced by a variety of species other than O2-, including oxidases such as xanthine oxidase, 1~ and appropriate controls should be performed. Cells are harvested, washed (as described above), and toluenized in 17 mM sodium pyrophosphate buffer, pH 8.3. A 0.2 ml sample of the aqueous toluenized cell phase is added to a tube of the same buffer containing 50/zM NBT and 78/zM NADH (freshly prepared). PMS (5 ~M final concentration) is added to initiate the reaction, and the rate of NBT reduction is followed at 560 nm. Boiled sample and excess SOD controls should always be run to ensure that the observed NBT reduction is 02dependent and that its inhibition by the sample is independent of heatstable species. It should be noted that D-amino-acid oxidases and some diaphorases can also reduce PMS so that if the toluenized cells contain significant quantities of these enzymes the initial flux of Oz- generated may be elevated. Measurement of Superoxide Dismutase in Individual Colonies
A method for determining the approximate SOD levels in each of numerous colonies on an agar plate can be of great value in isolating SOD structural and regulatory mutants and distinguishing SOD-rich from SODto M. Nishikimi, N. A. Rao, and K. Yagi, Biochem. Biophys. Res. Commun. 46, 849 (1972). u C. Beauchamp and I. Fridovich, Anal. Biochem. 44, 276 (1971).
S O D ASSAYS IN WHOLE BACTERIAL CELLS
poor species in mixed populations. The only suitable method developed at present is that of Schiavone and Hassan for Escherichia coli, 12an abbreviated version of which follows. Method. Colonies of E. coli are picked, grown overnight on LB agar plates, and blot-transferred to a Whatman #1 filter paper disk with an index mark. Replica plating onto the paper from dilution plates having scattered, well defined colonies can also be done. The paper disks are inverted and placed in 0.5 ml of a I mg/ml lysozyme solution for 30 min and then transferred to a desiccator having a chloroform atmosphere for further lysis (30 rain). The disks are frozen (10 min) and thawed 3 times, then transferred (colony side up) to plates containing 1% agar, 50 mM phosphate, and 0.1 mM EDTA, which are incubated for 3 hr at room temperature. The incubation allows diffusion of the released cytoplasmic enzymes, including SOD, into the agar. There should be no syneresis (free) water on the plates as this will cause blurting of the zones. The disks are removed from the plates, and 15 ml of 0.55 mM NBT, 66 /.~M riboflavin, 0.1% TEMED (N,N,N',N'-tetramethylethylenediamine), 1% agar, 50 mM phosphate, and 0.1 mM EDTA at 55° are overlaid on the plates in low light. After a further 3-hr incubation in the dark, the plates are exposed to even cool-white fluorescent until there is full development of the deep blue monoformazan color from the NBT (510 min). Areas containing SOD will block the O2--dependent production of color and will remain pale or achromatic. The assay is sufficiently sensitive to detect 0.05/zg pure SOD (about 0.16 units). This protocol, especially the cell lysis steps, may require modification depending on the cell type employed. Riboflavin Riboflavin* + T E M E D Riboflavin 7 + 02 O C + NBT (yellow)
) riboflavin* > riboflavin ~ + TEMEDox ) riboflavin + 02) 02 + monoformazan (deep blue)
(3) (4) (5)
Comments. Use 5-50 mM EDTA with the lysozyme in the absence of divalent cations. EDTA permeabilizes the outer membranes of many gram-negative bacteria, improving access of lysozyme to its substrate, the peptidoglycan wall. Placing of the freeze-thaw step before lysozyme treatment may also improve access of the lysozyme to the wall polymers. Since chloroform has been reported to release periplasmic, but not cytoplasmic, enzymes from E. coli, 13and toluene to release cytoplasmic proteins including SOD, 1-5 replacement of chloroform with toluene should improve results. z2 j. R. Schiavone and H. M. Hassan, Anal. Biochem. 168, 455 (1988). 13 G. F.-L. Ames, C. Prody, and S. Kustu, J. Bacteriol. 160, 1181 (1984).
ASSAY OF FORMATION OR REMOVAL OF OXYGEN RADICALS
For large numbers of small-volume cell suspensions, use of a micromultiweU filter suction apparatus (e.g., Minifold I of Schleicher and Schuell, Keene, NH) can produce sheets of paper with large numbers of precise spots containing known numbers of washed cells. Use of a 96-well microtiter plate format and dimensions for the paper-bound cells would allow subsequent rapid reading and photometric quantitation in a conventional microtiter plate reader. Toluene-mediated release of active enzymes from yeasts has also been reported.~ Thus, unicellular algae and fungi, as well as distinct colonies or clones of cells from plants or animals adherent on a solid surface should be amenable to SOD quantitation by the above methods.
 S u p e r o x i d e D i s m u t a s e M i m i c P r e p a r e d f r o m Desferrioxamine and Manganese Dioxide
By WAYNE F. BEYER, JR., and IRWIN FRIDOVICH Introduction Superoxide dismutases (SODs) are a family of metaUoenzymes which provide a defense against one aspect of the toxicity of dioxygen, by catalyzing the conversion of 02- to H202 plus O2)-3 Low molecular weight mimics of SOD might be very useful, both as antioxidants and as pharmaceutical agents, and have been vigorously sought. Manganese or copper ions, either free or complexed, are efficient catalysts of the dismutation reaction. SOD mimics based on complexed copper suffer from problems which include insolubility in the aqueous buffer systems, instability in the presence of serum proteins, and the possibility that copper, once freed from the complexing agent, might catalyze hydroxyl radical formation. In contrast, manganese complexes are stable in the presence of serum proteins and are unable to catalyze hydroxyl radical formation. We present a brief description of the preparation and properties of a soluble green complex prepared from desferrioxamine and manganese dioxide (MnO2) 4 which can catalyze the dismutation of 02- in oitro and which could prot j. V. Bannister, W. H. Bannister, and G. Rotilio, CRC Crit. Reu. Biochern. 22, 111 (1987). 2 I. Fridovich, Arch. Biochem. Biophys. 247, 1 (1986). 3 I. Fridovich, Ado. Enzymol. $8, 61 (1986). 4 D. Darr, K. A. Zarilla, and I. Fridovich, Arch. Biochern. Biophys. 258, 351 (1987).
METHODS IN ENZYMOLOGY,VOL. 186
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.