[1 81 M E A S U R E M E N T OF EPR-DETECTABLE IRON 173

[18] M e a s u r e m e n t of "Free" or Electron Paramagnet ic Resonance-Detec tab le Iron in Whole Yeast Cells

as Indica tor of Superoxide St ress

By CHANDRA SRINIVASAN and EDITH BUTLER GRALLA

Introduction

Yeast, like most eukaryotes, contain manganese superoxide dismutase (MnSOD; product of the S O D 2 gene) in the mitochondria and copper, zinc super- oxide dismutase (Cu,ZnSOD; product of the SOD1 gene) in the cytoplasm, mito- chondrial intermembrane space, and nucleus. There is no evidence of a separate extracellular SOD in yeast, as there is in mammals. Strains of S a c c h a r o m y c e s

cerevis iae that lack either or both superoxide dismutases have been available for many years and have provided much useful information. Experiments in our laboratory have highlighted the important roles of these two enzymes in aging (stationary-phase survival) and have helped define the cellular sources of super- oxide and the differing roles of the two enzymesJ -3 Selecting genetic suppres- sors of the s o d A mutant phenotypes has led to important discoveries relating to metabolism of copper and manganese, 4-8 and, more recently, iron. 9A° We discov-

ered that excess superoxide in the cell (due either to the absence of SOD in the s o d A mutants or in wild-type cells treated with the redox cycling drug paraquat) leads to accumulation of a form of iron that is detectable by electron paramag- netic resonance (EPR) spectroscopy at g = 4.3 (see below). 1° In this article we summarize the methods for growing the s o d A yeast strains and for analyzing their "free" iron content by Fe(III) EPR. It should be noted that in vivo there in no such thing as free i ron - - i t would certainly be bound to someth ing- - so we use the term "free" in quotes to denote iron that is loosely bound and accessible to chelator, or,

t V. D. Longo, E. B. Gralla, and J. S. Valentine, J. Biol. Chem. 271, 12275 (1996). 2 V. D. Longo, L. M. Ellerby, D. E. Bredesen, J. S. Valentine, and E. B. Gralla, J, Cell Biol. 137, 1581

(1997). 3 V. D. Longo, L. L. Liou, J. S. Valentine, and E. B. Gralla, Arch. Biochem. Biophys. 365, 131 (1999). 4 X. F. Liu and V. C. Culotta, Mol. Cell. Biol. 14, 7037 (1994). 5 X. F. Liu and V. C. Culotta, J. Biol. Chem. 274, 4863 (1999). 6 S. J. Lin and V. C. Culotta, MoL Cell, Biol. 16, 6303 (1996). 7 S. J. Lin and V. C. Culotta, Proc. Natl. Acad. Sci. U.S.A. 92, 3784 (1995). 8 V. C. Culotta, Metal Ions BioL Syst. 37, 35 (2000). 9 j. Strain, C. R. Lorenz, J. Bode, S. Garland, G. A. Smolen, D. T. Ta, L. E. Vickery, and V. C. Culotta,

J. BioL Chem. 273, 31138 (1998). 10 C. Srinivasan, A. Liba, J. A. Imlay, J. S. Valentine, and E. B. Gralla, J. Biol. Chem. 275, 29187

(200o).

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

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

174 MUTANTS, KNOCKOUTS, TRANSGENICS [ 18]

in the case of yeast, already existing as Fe(III) in a high-spin rhombic form, but not bound to enzyme active sites or to heme.

Fe(III) EPR spectra that are useful in biological systems fall into three g value classes: those with features in the g = 6 region for heme iron, those with three g features (x, y, z) clustered around g = 2 for low-spin iron species, and those at g = 4.3 for nonheme high-spin iron. 11 (g defines the position of the signal and the g value = hv/flH, where h is Planck's constant, v is the frequency, fl is the Bohr magneton, and H is the external magnetic field at resonance.) The signal at g = 4.3 is characteristic of ferric iron in a high-spin rhombic complex, and is the one we are concerned with here. Desferrioxamine-bound iron gives a signal at this g value, as do ferric citrate, other simple chelates, and oxidized [Fe(III)] transferrin. H is about 1550 G for the Fe(llI) EPR signal under our conditions. Liquid nitrogen temperatures are needed for detecting the Fe(III) EPR signal at g = 4.3 and liquid helium temperatures are required for the other two g regions. The ferrous form of iron cannot be detected in biological systems, as the signal is too broad.

The Fe(III) EPR signal at g --- 4.3 in the presence and absence of desferriox- amine has been monitored as an indication of the "free," or loosely bound iron in several organisms. In most organisms, the addition of desferrioxamine is necessary to observe the Fe(III) EPR signal at g --- 4.3, indicating that the "free" iron present is in the Fe(II) state and can be detected only after chelation and conversion to Fe(III) by this avid ligand. This is true in Escherichia coli le as well as in some mammalian systems, where a pool of chelatable or "labile" iron has been demonstrated that can be detected by EPR after treatment with desferrioxamine t 3 or by fluorescence methods with other chelators such as calcein. 14-16 The mammalian pool is thought to be the iron that is sensed by the iron regulatory protein (IRP), which binds a specific motif in mRNA (IRE) to regulate the synthesis of ferritin and transferrin receptor in response to changes in iron availability. 17 In E. coli, the chelatable iron was shown to be greatly increased by superoxide stress, that is, in mutants lacking superoxide dismutase (sodA sodB), lz18 This increase may be attributable to release of iron from superoxide-sensitive iron-sulfur cluster proteins such as aconitase, as was originally proposed by Liochev and Fridovich. 19

11M. C. R. Symons and J. M. C. Gutteridge, "Free Radicals and Iron: Chemistry, Biology, and Medicine." Oxford University Press, Oxford, 1998.

12 K. Keyer and J. A. Imlay, Proc. Natl. Acad. Sci. U.S.A. 93, 13635 (1996). 13 A. V. Kozlov, A. Bini, D. Gallesi, F. Giovannini, A. Iannone, A. Masini, E. Meletti, and A. Tomasi,

Biometals 9, 98 (1996). 14 Z. I. Cabantchik, H. Glickstein, P. Milgram, and W. Breuer, Anal Biochem. 233, 221 (1996). 15 W. Breuer, S. Epsztejn, and Z. I. Cabantchik, FEBS Lett. 382, 304 (1996). 16 S. Epsztejn, O. Kakhlon, H. Glickstein, W. Breuer, and I. Cabantchik, Anal. Biochem. 248, 31 (1997). 17 W. Breuer, S. Epsztejn, and I. Cabantchik, J. Biol. Chem. 270, 24209 (1995). 18 K. Keyer and J. A. Imlay, J. Biol. Chem. 272, 27652 (1997).

[ 18 ] MEASUREMENT OF EPR-DETECTABLE IRON 175

In collaboration with the laboratory of J. A. Imlay, we developed a similar EPR method for yeast and found that superoxide stress causes accumulation of EPR- detectable iron in yeast as well, but, surprisingly, the iron is already in the Fe(III) state, and the bulk of it can be detected without the addition of desferrioxamine. 1°

The elevation of EPR-detectable or "free" iron in yeast correlates with condi- tions of elevated superoxide, and thus is a symptom of superoxide stress. In addi- tion, it may cause some of the detrimental effects of the lack of SOD in sod mutant strains, because "free" iron could catalyze many destructive oxidation reactions via Fenton chemistry) 9 The Fe(III) EPR method we describe can provide an in vivo assessment of oxidative stress status in yeast and E. coli, and we are currently testing whether this is true for other, more complex, organisms as well. One im- portant advantage of this method is that the analysis is performed on whole cells, eliminating uncertainties about the effects of cell lysis on the results. Processing of samples is minimal, consisting only of a short incubation in desferrioxamine (which often can be omitted) and some quick washing steps. Prepared samples are stored in EPR tubes at - 7 0 ° until they can be analyzed. This article outlines culture methods for these strains and describes how the Fe(III) EPR samples are prepared and analyzed.

M e t h o d s

Growth of sodA Yeast Strains

Saccharomyces cerevisiae lacking Cu,ZnSOD (sodl A ) or MnSOD (sod2A ) and sodlA/sod2A double knockouts have been constructed in our laboratory in the DBY746 (EG103) background and have been widely used. 2°m The indivi- dual sod knockout strains in the BY4741 genetic background can be purchased from Research Genetics (Huntsville, AL). Phenotypes of the mutants in either backgrounds are similar.

Media. These strains are grown in normal yeast media--YP-based rich media and SC defined medium22--with a few modifications 1 as described below. Because we have noticed some effects of the medium on the growth of the sod mutants, we are including some details about our medium preparation. YPD medium is the standard yeast culture medium and consists of 1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) dextrose (glucose). For plates, 2% (w/v) Difco (Detroit, MI) Bacto-agar is included. Other carbon sources can be substituted for the dextrose,

19 S. I. Liochev and I. Fridovich, Free Radic. Biol. Med. 16, 29 (1994). 2o E. B. Gralla and D. J. Kosman, Adv. Genet. 30, 251 (1992). 21 E. B. Gralla, in "Oxidative Stress and the Molecular Biology of Antioxidant Defenses" (J. Scandalios,

ed.), p. 495. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1997. 22 C. Kaiser, S. Michaelis, and A. Mitchell, "Methods in Yeast Genetics." Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, New York, 1994.

176 MUTANTS, KNOCKOUTS, TRANSGENICS [ 18]

for example, 3% (v/v) glycerol (making YPG), 2% (v/v) ethanol (YPE), 2% (w/v) galactose (YPGal), or a combination of glycerol and ethanol (YPGE). To avoid darkening (burning) of the carbon source when it is autoclaved in the medium, it is best to sterilize the carbon source separately, as a 10× solution, either by autoclaving or filtering through a 0.22-#m pore size filter, and then add it to the YP mix (made at 0.9 volume) when it has cooled somewhat. SC medium (synthetic complete, or defined medium) is based on Difco yeast nitrogen base (YNB) [per liter: 1.8 g of YNB without amino acids or ammonium sulfate, 5 g of ammonium sulfate, 1.4 g of NaHePO4, dry amino acid mix (described below)]. Ammonium sulfate is the nitrogen source; a carbon source [usually 2% (w/v) dextrose, but others are fine, too] is sterilized separately and added as a 10× solution. Amino acids, adenine, and uracil are added to the medium as a dry mix to improve growth and compensate for auxotrophic markers the strain may carry. The sod1 mutants grow better with increased amounts of certain amino acids, so we use the following amounts for all yeast culture (in mg/liter): Ade, 80; Ura, 80; Trp, 80; His-HC1, 80; Arg-HCI, 40; Met, 80; Tyr, 40; Leu, 120; lie, 60; Lys-HC1, 60; Phe, 60; Glu, 100; Asp, 100; Val, 150; Thr 200; Ser, 400. It is convenient to make a dry mix of amino acids with enough for 10 liters of medium. The amino acids are crushed and mixed with a mortar and pestle, weighed out as needed (1.73 g/liter of the complete mix), and added to the bulk SD medium before autoclaving. Dropout mixes can be made by leaving out the desired component and adjusting the amount to be added. Additional information on various medium preparations and basic techniques used in yeast culture are described in several places. 22-24

Because the sodlA strains in particular are weakened and oxygen sensitive, it is easy inadvertently to select second-site suppressors during growth and/or during storage on plates. In addition, both sodlA and sod2A mutant strains die quickly on plates. To avoid problems and increase reproducibility each experiment is started with freshly streaked frozen stocks. Strains carrying the sodlA mutation are revived from frozen cultures on YPD plates in a microaerophilic atmosphere, using BBL Campy pouches (Becton Dickinson Microbiology Systems, Sparks, MD). These bags reduce oxygen to 5 to 10% and increase the CO2 levels, and they improve growth of sodl A strains without the added complications and metabolic alterations that true anaerobiosis induces. The culture process for a typical EPR experiment is outlined below.

1. Day 1. Streakcells from frozen stocks onto YPD agar plates to obtain single colonies. Place the soda yeast plates in a BBL Campy pouch (Becton Dickinson)

23 C. Guthrie and G. R. Fink, (eds.), "Guide to Yeast Genetics and Molecular Biology," Vol. 194. Academic Press, San Diego, California, 1991.

24 E M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, in "Current Protocols in Molecular Biology," Chap. 13. John Wiley & Sons, New York, 1998.

[ 18] MEASUREMENT OF EPR-DETECTABLE IRON 177

after adding the liquid activating reagent. Incubate the plates at 30 ° for 2 to 3 days, until colonies are visible on all the plates.

2. Day 3. Inoculate starter cultures. Starter cultures are grown in 16 x 150 mm tubes in 4 to 5 ml of the medium in which the experiment will be conducted (usually SDC, but SGC, YPD, or various SD dropout media also can be used). Remove the plates from the Campy pouch. Using a sterile wire loop, toothpick, or applicator stick, transfer a single colony into the liquid medium and grow overnight (12-18 hr) at 30 ° with shaking at 220 rpm. It is best to pick an average-sized colony from the sod mutant plates--large ones are likely to be suppressors. Make duplicate cultures for each strain, using a different colony for each overnight culture. These growth conditions are not fully aerated, so the mutant strains are able to grow well and are not overly stressed. It is important not to overgrow the overnights as soda strains begin to lose viability on entry into stationary phase,

3 Day 4. Start experimental cultures. The density of each overnight culture is measured by diluting 50/xl of (fully resuspended) culture to 1 ml in distilled water and reading the optical density at 600 nm (OD600) in an ultraviolet-visible wavelength (UV-Vis) spectrophotometer. Spectrophotometer readings should be below 0.5, as the correlation between cell density and "absorbance" is not linear above this limit, so cells should be diluted until a low enough reading is obtained. In our hands, and OD600 of 1 is equivalent to 1 × 107 cells/ml. For SDC growth the cells usually reach a maximum density of between 5 × 107 and I0 x 107 cells/ml, or an OD600 of 5 to 10; for YPD growth it can be much higher (20 x 107 to 30 x 107 cells/ml). The sodlA mutants generally grow to a lower final density than the wild type. The amount of overnight culture needed to obtain a starting OD600 of 0.1 (106 cells/ml) in 50 ml of medium is calculated and 250-ml culture flasks containing 50 ml of medium are inoculated with the calculated amount of each overnight. Flasks are placed in an incubator at 30 °, with shaking at 220 rpm. Other flask sizes can be used, but it is important that the ratio of flask volume to medium volume be at least 5 : 1 to maintain good aeration. Good aeration for the experimental cultures is essential as the phenotype of the mutants is expressed only under aerobic conditions. Typically, cells are harvested after 24 or 72 hr of growth and prepared for EPR as described below. If log-phase cells are desired a similar protocol is used, but much larger volumes will be needed as cells should be harvested at an OD600 of 1 or below.

Electron Paramagnetic Resonance Sample Preparation

The Fe(III) EPR method we are using currently was developed for E. coli by Keyer and Imlay 12,18 and modified for our yeast system. The original procedure relied on the ability of desferrioxamine to enter the cell and chelate loosely bound iron or iron not specifically bound to proteins or other high-affinity or biologically protected sites, converting it to Fe(III) and rendering it detectable by EPR at

178 MUTANTS, KNOCKOUTS, TRANSGENICS [18]

g ---- 4.3. In E. coli the desferrioxamine was essential to detect a signal, indicating the most loosely bound iron was present as Fe(II) in that organism. In yeast, on the other hand, the iron is detectable whether or not the desferrioxamine was added, indicating that the iron is already in the Fe(III) state, l°

Day 5 or 7. Typically, cultures are grown for 24 or 72 hr because at these times we see the largest differences in EPR-detectable iron between wild type and sod mutants. At 24 hr cultures in SDC are saturated, but are still transitioning out of active growth; by 72 hr they are entering stationary phase and are set up for long-term survival without growth. The most consistent results are obtained with 72-hr cultures.

After the required growth period, the OD600 is recorded for each culture as described above and the cell harvest procedure is begun. From this point on it is important to work as quickly as possible. Each culture is processed in duplicate. Transfer 10-ml aliquots of culture to each of two centrifuge tubes. (Disposable 50-ml conical screw-cap centrifuge tubes work well.) Spin at 4 ° for 5 min at 4000 rpm. Discard the supernatant, resuspend the pellets in 9 ml of YP or SC medium (no sugar added), and transfer the contents into 50-ml sterile Erlenmeyer flasks. To one flask add 1 ml of 0.2 M desferrioxamine and to the other add 1 ml of buffer. [Make a fresh stock of 0.2 M stock solution of desferrioxamine mesylate salt (Sigma/Aldrich, St. Louis, MO) in water and adjust to pH 8 with 1 M KOH.] Incubate both samples at 30 ° with shaking for 12-14 rain. Transfer the samples to centrifuge tubes and spin at 4 ° for 5 min at 4000 rpm. Discard the supernatant and suspend the cell pellets in 10 ml of cold 20 mM Tris-HC1, pH 7.4, by pipet- ring up and down a few times. Centrifuge the suspension for 5 min at 4000 rpm and 4 °. Carefully remove the supernatant without disturbing the cell pellet. Add 200/zl of 20 mM Tris-HC1, pH 7.4, containing 10% (v/v) glycerol to the pel- let and resuspend well. Measure and record the total volume of the suspension. Transfer exactly 200 #1 of this sample to a 4-mm diameter EPR tube (Wilmad Glass, Buena, NJ), making sure that the cell suspension reaches the bottom of the tube. Immediately freeze samples on dry ice, and store at - 7 0 ° until EPR mea- surements can be performed. The volume of culture used, the final OD60o of the culture, and the total volume of EPR sample before transferring of the 200 #1 to the EPR tube must be recorded for calculation purposes.

Whole-Cell Low-Temperature Fe(III) Electron Paramagnetic Resonance

EPR spectra of the "free" and desferrioxamine-chelated iron(III) are measured by Fe(III) EPR. We use a Bruker (Billerica, MA) X-band spectrometer (model ESP300E), although other instruments can be used as well. Samples are main- tained at - 125 ° during the recording of the spectra, using a finger Dewar (Wilmad Glass) filled with liquid nitrogen. Before running samples each day, the spectrum

[18] MEASUREMENT OF EPR-DETECTABLE IRON 179

of an iron standard of known concentration (31/zM in our case) is recorded and the EPR instrument is tuned and the position of the finger Dewar adjusted to maximize the signal intensity. The iron standard is prepared by taking a solu- tion of ferric sulfate of known concentration and diluting it in 20 mM Tris-HC1 buffer., pH 7.4, containing 1 mM desferrioxamine to make a solution approximately 0.1 mM in Fe(III). The exact concentration of this solution is then determined by measuring the absorbance of an aliquot of this solution at 420 nm (extinction coefficient = 2865 M -1 cm-l). This concentrated solution is further diluted in 20 mM Tris-HCl-10% (v/v) glycerol, to make a standard of the desired concentra- tion. The same iron standard can be used over and over again by storing at - 7 0 ° . It is a good idea to acquire spectra for this standard several times over the course of the day to test the performance of the instrument. To compare the values obtained for different samples with the standard it is important to keep all EPR parameters unchanged, including factors such as number of scans and instrument gain. After inserting each sample, the instrument is autotuned and acquisition is started.

Parameters used for whole cell low-temperature Fe(III) EPR are as follows: frequency, 9.27 GHz; center field, 1500 G; sweep width, 500 G; microwave power, 20 mW; attenuation, 10 dB; modulation amplitude, 20.0 G; modulation frequency, 100 kHz; receiver gain, 1 × 105; sweep time, 41.9 sec; time constant, 20.48 msec; conversion time, 10.24 msec; resolution, 4096 points; number of scans, 16.

Electron Paramagnetic Resonance Data Processing and Calculations

EPR data processing is done with the Bruker WinEPR program. Spectra of samples and the iron standard are filtered to decrease noise and the baseline is corrected. The signal at g = 4.3 is quantitated by double integration, using the same software, and the double integral (DI) value is noted.

The total free iron (in moles) in the resuspended sample is calculated as follows:

( DIx [Fe]std "Free" Fe = \ ~ j Vresusp

where DIx is the double integral of the sample, DI~ta is the double integral of the standard, and Vresusp is the volume of the whole sample (of which 200/zl is transferred to the EPR tube).

The number of cells in the resuspended sample is calculated from the OD600 of the culture when it is harvested, the volume in ml of culture used (Vcult), and the conversion factor 1 × 107 cells per OD of 1.

No. of cells = OD60o × 107 × Vcult

Substituting these numbers in the following equation gives the intracellular concentration of EPR-detectable iron ([free Fe]). We assume that there is little or

180 MUTANTS, KNOCKOUTS, TRANSGENICS [ 19]

no iron in the suspension buffer, because of the washing steps, so any iron in the sample is located inside the cells. The volume of a single haploid yeast cell is 70 fl; that of a single diploid cell is about 150 1. 23

free Fe/no. of cells [F ree Fe]intracellula r =

single-cell volume

We hope that this method proves useful for studies of superoxide stress, other kinds of oxidative stress, and iron metabolism.

A c k n o w l e d g m e n t s

We express our sincere gratitude to Dr. James A. Imlay for helping us initiate these studies of the yeast system, and to Drs. Barney Bales and Miroslav Peric for their help with EPR. This research was supported by NIH Grant DK46828 to Dr. Joan S. Valentine.

[19] Transgenic Superoxide Dismutase Overproducer: Murine

B y SERGE PRZEDBORSKI, ~ R N I C E JACKSON-LEWIS, DAVID SULZER,

ALl NAINI, NORMA ROMERO, CAIPING CHEN, and JULIA ARIAS

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

Reactive oxygen species (ROS), such as superoxide and hydroxyl radicals, are produced constantly during normal cellular metabolism, and presumably even more so in a number of pathological conditions. Defense mechanisms, however, exist to limit the levels of ROS and the damage they inflict on cellular components such as lipids, proteins, and DNA. It has been hypothesized that the finely tuned balance between the production of ROS and their destruction is skewed in a number of diseases and in normal aging, resulting in oxidative damage that leads to severe cellular dysfunction and, ultimately, to cell death.1

Among the many ROS-scavenging enzymes, superoxide dismutase (SOD) has received the lion's share of attention not only because of its key role in ROS metabolism, but also because mutations in a member of the SOD family can cause an inherited form of the paralytic disorder amyotrophic lateral sclerosis (ALS). SOD comes in three isoforms: two copper, zinc SODs (cytosolic SOD1 and extracellular SOD3) and a manganese SOD (mitochondrial SOD2). 2 All three

I s . Przedborski and V. Jackson-Lewis, in "Free Radicals in Brain Pathophysiology" (G. Poli, E. Cadenas, and L. Packer, eds.), p. 273. Marcel Dekker, New York, 2000.

2 I. Fridovich, Annu. Rev. Biochem. 64, 97 (1995).

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