Wheat Superoxide Dismutase Isoenzymes

Wheat Superoxide Dismutase Isoenzymes

Journal of Cereal Science 23 (1996) 93–101 Wheat Superoxide Dismutase Isoenzymes D. S. Robinson∗‡, J. K. Donnelly∗, S. M. Lawlor∗, P. J. Frazier† and...

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Journal of Cereal Science 23 (1996) 93–101

Wheat Superoxide Dismutase Isoenzymes D. S. Robinson∗‡, J. K. Donnelly∗, S. M. Lawlor∗, P. J. Frazier† and N. W. R. Daniels† ∗University of Leeds, Procter Department of Food Science, Leeds LS2 9JT, U.K. and †Dalgety Food Technology Centre, Station Road, Cambridge CB1 2JN, U.K. Received 13 February 1995

ABSTRACT Evidence was obtained indicating that the superoxide dismutase (SOD) A and B isoenzymes of the wheat cultivar Tonic are of the Cu/Zn type, whereas the SOD–C group are similar to manganese containing dismutases. Heat inactivation plots showed that the enzymic activity in crude extracts was relatively stable up to 50°C. The thermodynamic parameters enthalpy, DH#, free energy, DG#, and entropy, DS#, were estimated for the thermal inactivation of extracted SOD activity and a purified SOD–C isoenzyme. The SOD-C isoenzyme was purified extensively and shown to contain two closely related tetrametic isozymes (pI6·0 and 6·1) of Mr 80 000. A Mr of 20 000 has been calculated for the subunits. The N-terminal amino acid sequence of the purified SOD–C could be aligned with that of the Mn–SOD enzyme of maize and showed 57% homology.  1996 Academic Press Limited

Keywords: superoxide dismutase, wheat, cereals, heat inactivation

and thus the more reactive OH· radical may be generated via the Fenton reaction:

INTRODUCTION Superoxide dismutase (SOD) is responsible for catalysing the dismutation of the superoxide radical to oxygen and hydrogen peroxide.

M(n−1)++H2O2uMn++OH·+OH−

202−+2H+uO2+H2O2

Active oxygen species are possible initiators of oxidative reactions in foods, which may include the oxidation of various food constituents such as lipids, vitamins and flavour compounds. Such reactions will bring about detrimental effects on the sensory and nutritional properties and possibly lead to the formation of toxic substances. In fresh foods, SOD is naturally present in biological materials as a series of discrete isoenzymes in various organelles. During food processing, which may include mechanical disruption, freezing, heat treatment and drying, the SOD isoenzymes may be wholly, partially or differentially inactivated, which may increase the susceptibility of food ingredients both to autoxidation and possibly enzyme catalysed oxidations. Michelson and Monad1 found the autoxidation of anchovy oil was delayed by the presence of SOD and, recently bovine and pea SOD were found to inhibit lipid autoxidation

By removal of superoxide the enzyme is able to prevent both redox reactions directly involving O2− and those where O2− may generate more reactive radicals. For example, superoxide may act as a metal ion reductant, Mn++O2−uO2+M(n−1)+

 : SOD=superoxide dismutase; Mr=relative mass; PAGE=polyacrylamide electrophoresis; NBT=nitroblue tetrazolium; TEMED= N’N’N’N’–tetramethylethylenediamine; EDTA=ethylenediamine tetraacetic acid; IEF=isoelectric focusing; DDTC=diethyldithiocarbamate; DEAE Sephadex= diethylamino Sephadex. ‡ To whom correspondence should be addressed. 0733–5210/96/010093+09 $12.00/0

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specifically2,3. Also, the elimination of the superoxide radical in foods may be achieved by the addition of SOD, for which a patent has been granted1. SOD is not currently used as a commercial additive to foods, firstly because its potentially beneficial role in foods is poorly understood, and secondly because a cheap and reliable commercial source is neither available nor approved for food use by regulatory authorities. Therefore, in order to exploit SOD fully as a food antioxidant it is important to study the occurrence of the SOD isoenzymes in foods, establish their thermostabilities and examine suitable sources as potential natural additives2–6. Isoenzymes can be distinguished by analytical isoelectric focusing (IEF) and polyacrylamide gel electrophoresis (PAGE), in which they appear as groups of close moving bands. Although the number of SOD isoenzymes discovered may vary depending on both the experimental procedures and the organs used, in plants there are usually two Cu/Zn-SOD isoenzymes and one Fe-SOD or/ and Mn-SOD isoenzyme present. The reason for the evolution of three types of SOD to catalyse the same specific dismutation of O2− has not been elucidated. Two Cu/Zn-SOD isoenzymes have been purified from wheat germ and one suspected Mn–SOD isoenzyme has been found by Beauchamp and Fridovich. Three groups of SOD isoenzymes of wheat can be separated electrophoretically by PAGE5. Each isoenzyme group shows multiple isoenzyme bands varying slightly in pI values as shown by isoelectric focusing. Owing to its structural characteristics and the location of the isoenzymes in wheat seeds6, MnSOD is thought to be associated with mitochondria and the two Cu/Zn isoenzymes with chloroplasts and cytoplasm. Jaaska5 monitored the isoenzyme profile of SOD in wheat, rye, goatgrass and triticale by PAGE, and the allo-enzyme variation by isoelectric focusing. Jaaska5 concluded that the isoenzymes were evolutionary rather than conservative, showing only rare cases of variation among the barley species. All wheat and rye species had one SOD-C band of closely similar electrophoretic mobility and isoelectric point (pI≈6·20). Cox et al.7 aimed to provide useful data for genetic and breeding studies by studying the isoelectric focusing patterns of SOD from 80 North American wheat cultivars. They found no detectable differences among the cultivar patterns, although the SOD bands were poorly defined.

In the present communication we report the purification, the thermostability and characterisation of a purified wheat SOD isoenzyme from the cultivar ‘Tonic’. MATERIALS Nitroblue tetrazolium (NBT), bovine serum albumin, riboflavin, N,N,N′,N′-tetramethylethylenediamine (TEMED), sodium cyanide and diethylenetriamine-pentacetic acid (DETAPAC), glycerol and ethylene glycol were supplied by Sigma Chemical Co. Ltd., Poole, Dorset. Superoxide dismutase (bovine), ethylenediamine tetraacetic acid (EDTA) and Triton X100 were obtained from Boehringer BCL, Lewes, Sussex. Ortho-dianisidine (3,3′-dimethoxybenzidine) was obtained from KochLight Laboratories, Colnbrook, Buckinghamshire. Ampholine carrier ampholytes for isoelectric focusing (IEF) and Polybuffer 74 were obtained from Pharmacia Ltd., Milton Keynes, Buckinghamshire. All other chemicals were obtained from BDH Ltd., Poole, Dorset, and were of AnalaRTM grade. Grain of the wheat cultivar, Tonic, was supplied by Dalgety Food Technology Centre, Cambridge. Samples (100 g) were milled at 10 s intervals for 1 min in a coffee mill. Polybuffer solutions were prepared with 10% (w/v) polybuffer 74, in (20%) w/v ethyleneglycol and then titrated to the required pH with 1  HCl. EXPERIMENTAL Assay of SOD A riboflavin-nitroblue tetrazolium assay was adapted from Beauchamp and Fridovich8. The photosensitive assay reagent contained 0·585 l riboflavin, 56 l NBT, 0·05% (w/v) TEMED, 0·01% (w/v) Triton X-100 and 1 m DETAPAC in 0·05  phosphate buffer, pH 7·8. Triton X-100 was added to the photochemical assay reagent to prevent the precipitation of formazan9. DETAPAC was added as it is thought to enhance the sensitivity of the NBT assay methods10. The solution, which was made up in subdued light, remained stable for one day at room temperature, and was kept in a foil covered automatic dispenser. The reaction mixture contained the photosensitive assay reagent (0·5 ml), extracts (up to 50 ll) and

Wheat superoxide dismutase enzymes

was made up to a total volume of 3 ml. Cuvettes were held in a 24-slot rack and interspaced with controls to allow correction for any uneven illumination along the length of the fluorescent tube. Illumination was for 6 min in a foil-lined light box with two 20 W tubes at room temperature. To provide homogeneous illumination, the fluorescent tubes were aged for 100 h before use. The inhibition of the NBT reduction was measured at 560 nm, and a linearised calibration plot was adopted11. SOD activity was expressed as lg equivalents of bovine SOD.

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Protein stains Gels were fixed in a solution containing 3·46% (w/v) sulphosalicylic acid and 11·5% (w/v) trichloroacetic acid. They were stained with 0·115% (w/v) Coomassie Brilliant Blue R250 in an aqueous solution of 25% (v/v) ethanol and 8% (v/v) acetic acid. Silver staining was as outlined in the Pharmacia PhastSystem development technique file number 210. Samples contained 1–5 ng of protein/ll of sample prior to electrophoresis for silver staining. Inhibitors of SOD activity

Heat treatment of the purified wheat SOD isoenzymes Aliquots that inhibited NBT reduction by 30–50% were pipetted into several thin walled glass tubes pre-equilibrated at the appropriate temperature. After heat treatment the contents were cooled quickly in ice-cold water and mixed with the assay reagent. Measurements were carried out in triplicate for each time/temperature combination. The SOD activity of the unheated extracts represented the initial (100%) activity from which the proportion (%) of the remaining activity after heating was calculated.

The following were used as inhibitors: 3 m sodium cyanide, 0·3  hydrogen peroxide 0·02  diethyldithiocarbamate (DDTC) and chloroform/ ethanol. Extract (30 ll) was applied to the IEF gel as the control alongside: (a) 5 ll of 30% (v/v) H2O2 in 25 ll of extract; (b) 15 ll of chloroform/ethanol (0·25 : 0·15) with 15 ll of extract; (c) 3 ll of 0·2  DDTC in 27 ll of extract; and (d) 3 ll of 0·3  NaCN in 27 ll of extract. The focused gel was stained for SOD activity. Preparation and extraction of SOD from wheat

Analytical isoelectric focusing (IEF)

Milling of wheat kernels Small samples (100 g) of wheat were milled at 10 s intervals for 1 min in the laboratory with a coffee mill to produce fresh whole flour.

Analytical IEF was carried out on a flat bed LKB system, with 5% polyacrylamide gels of 0·5 mm or 0·75 mm thickness. The pH ranges used were pH 4·5–6, pH 5–7 and pH 4–8. For a 0·5 mm gel containing ampholines (pI 4–6·5), electrophoresis was carried out at 20 W with limits of 20 mA and 1600 V for at least 2 h. After electrophoresis, the pH gradient was determined after cutting the gel into 5 mm pieces. Staining for SOD activity was as described by Beauchamp and Fridovich8. Reagent A was a stable aqueous solution of 1·2 m NBT, and was kept in the dark at 4 °C. Reagent B was 2·9×10−5  riboflavin and 0·28  TEMED in phosphate/DETAPAC buffer, which was always freshly prepared. The focused gel was soaked in reagent A followed by immersion in reagent B for 15 min in the dark. The gels were then illuminated for up to 30 min with a 30 W fluorescent tube. SOD isoenzymes were observed as white unstained zones against a blue background.

Ammonium sulphate fractionation Crude extracts of the freshly milled wheat were prepared by stirring with 0·05  phosphate buffer, pH 7·8 containing 1 m EDTA for 2 h at room temperature, followed by filtration through muslin and centrifugation at 17 000 g for 15 min. The supernatant was dialysed against 100-fold volume of the above buffer at 4 °C for a minimum of 2 h. Solid ammonium sulphate was slowly added to a stirred solution of crude extract to give 40% saturation and equilibrated for 30 min at room temperature. Centrifugation was at 17 500 g for 20 min. A further addition of ammonium sulphate was made to the supernatant to give 70% saturation and equilibrated for 30 min. The collected precipitate was redissolved in 0·05  Tris-HCl buffer, pH 7·2 and dialysed first against distilled water overnight and, second, against a 20-fold volume of 0·02  Tris-HCl, pH 7·2 (20 volumes) prior to further purification.

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Anion exchange chromatography

DEAE-Sephadex A50 The extract (100 ml) was mixed with pre-swollen anion exchange resin (5 g), made up to 500 ml with 0·02  Tris-HCl, pH 7·2 and equilibrated at 4 °C. The resin and buffer were separated by Whatman No. 4 paper and the resin washed with 0·02  Tris buffer (1 l). The retained anion exchanger containing bound SOD was resuspended in 0·02  Tris-HCl, pH 7·2 (400 ml) containing 0·4  NaCl, and equilibrated for 15 min at 4 °C. The gel slurry was filtered and eluted with the same buffer. The effluent (500 ml) was concentrated to 50 ml by ultrafiltration on an amicon PM30 membrane with an MW cut off of 30 000. Q-Sepharose A Pharmacia XK column (26 mm×100 mm) was packed with pre-swollen anion exchanger and equilibrated with several column volumes of 0·02  Tris-HCl, pH 7·8. A concentrated sample (50 ml) from the preceding batch separation with DEAESephadex A50 was dialysed against 0·02  TrisHCl, pH 7·8 and applied to the anion exchange column. Unbound proteins were eluted with the same the Tris buffer (100 ml) prior to further elution with a linear NaCl gradient (0–0·4  NaCl) at a flow rate of 3·5 ml/min. The fractions (5 ml) were assayed for SOD activity and analysed by isoelectric focusing. Protein was estimated by absorbance at 280 nm. Preparative isoelectric focusing The pooled fractions obtained by chromatography on Q-Sepharose were dialysed against 0·05  phosphate buffer, pH 7·8 containing 1% (w/v) glycine. The sample was concentrated to 2 ml and made up to 3 ml with Ampholine solution (0·5%). The carrier ampholytes used had a pH range of either 4–6·5 or 5–7. The gel (210×110 mm) was prefocused for 90–120 min until the settings were approximately 8 W, (4 mA and 2000 V). Focusing, which occurred over the length of the gel, required 16 W with limits of 16 mA and 2000 V for 4 h at 4 °C. A sheet of absorbant paper was applied to the surface of the focused gel and, from it, zones of SOD activity were detected. The separated isoenzymes were recovered by means of a 30sectioned metal grid inserted into the gel. Eluted fractions were assayed for SOD activity and protein, and assessed by analytical IEF for both SOD

isoenzymes. The recovered SOD containing fractions were stored at −18 °C in 25% (v/v) glycerol. Chromatofocusing A Mono-P column (Pharmacia) was equilibrated with the start buffer (pH 6·4) and sample (5 ml) applied to the column via a Pharmacia superloop. Polybuffers (pH range 4·0–5), containing 20% (v/ v) ethyleneglycol to aid the solubility of wheat SOD-C isoenzyme, were passed through the column at a flow rate of 0·5 ml/min. Low back pressure was maintained by prior filtering of buffers and samples through a 20 lm Millipore membrane and thorough degassing of solutions. Fractions (1 ml) were collected and held at 0 °C before assaying for SOD activity. The purified isoenzymes were stored in 25% (v/v) glycerol at −18 °C. Criteria of purity Samples (1 ll) of the purified isoenzymes were subjected to PhastSystem analytical IEF on a PhastGel pH range 4–6·5. After focusing, the gels were stained for protein with silver. The pH gradient profile was determined by using the low pI calibration kit containing a mixture of 5 standard proteins covering the pI range of 4·15–6·55. An aliquot of the standard protein mixture was run alongside the SOD isoenzyme samples, and the Rf values directly measured from the gel, to produce a pI calibration curve. The Rf values of the purified SOD isoenzymes were used to estimate the pI values from the calibration curve. Determination of relative molecular mass (Mr) Mrs were determined by two methods. In the first gel filtration a column (16 mm×620 mm) of Sephadex G-100 was used. Calibration was with purified protein standards (Pharmacia calibration kit). The second was under non-denaturing conditions using the PhastSystem. Samples of purified isoenzymes (1–4 ll) were subjected to electrophoresis on an homogeneous polyacrylamide media (12·5% w/v) preceded with a stacking zone. Proteins of known Mr were used as standards. Protein was detected using a silver stain as outlined in the PhastSystem separation technique file No. 121 (Pharmacia LKB).

Wheat superoxide dismutase enzymes

Figure 1 Isoelectric focusing of superoxide dismutases in crude extracts of whole meal from the wheat cultivar Tonic. The main isoenzyme groups are identified at regions A, B and C.

Subunit Mrs of SOD isoenzymes were determined by SDS–PAGE using the PhastSystem12. RESULTS AND DISCUSSION Isoelectric focusing resolved the SOD isoenzymes into three distinct groups (A, B and C) according to their isoelectric point (Fig. 1). The pI values of the major focused isoenzyme groups, estimated from the pH gradient, were 5·1, 5·2–5·3 and 6·0–6·1 for SOD-A, SOD-B1, B2, B3 and SODC, respectively. It is possible that the multiple isoenzymes for the SOD-B and SOD-C groups might be variants due to small changes in amino acid composition. Specific tests using the selective inhibitors NaCN, DDTC, H2O2 and chloroform-ethanol with both PAGE and IEF gels have indicated that the wheat isoenzymes SOD-A and B were of the Cn-Zn type13,14. The SOD-C isoenzymes, of which there are at least four, were inhibited only by the chloroform-ethanol treatment, which is characteristic of the manganese containing group of superoxide dismutases15. The heat inactivation of the crude and purified SOD isoenzymes followed first order kinetics over the limited temperatures range of 50–70 °C (Figs 2, 3). Heat activation parameters (Ea) determined using Arrhenius plots (Fig. 4) for enzyme inactivation were 160 kJ/mol and 82 kJ/mol for the

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SOD activity in the crude extract and for the purified SOD, respectively. For the purified preparation our calculated Ea of 82 kJ/mol is lower than that for the SOD activity present in the crude extract indicating that, for the purified isoenzyme, substantial protein unfolding may not be the rate limiting step and that simple chemical release of the bound metal may have been responsible for the observed inactivation. However, this conclusion depends on the validity of the assumed straight line relationship in the Arrhenius plot (Fig. 4). Using the Eyring equation DG#=RTln(k∗h/kT), where k∗, k and h are the rate constant, the Boltzman and Planck constants, respectively, values for DG# (the apparent Gibbs free energy of activation at 60 °C) of 100 kJ/mol and 102 kJ/mol for irreversible enzyme inactivation were obtained for the crude and purified SOD activity, respectively. The enthalpy and entropy values of heat inactivation at 60 °C of 156 kJ/mol and 171 J/mol/K, respectively, were determined from DH#=Ea−RT and DS#=(DH#−DG#)/T. H and S for the purified SOD were 79 kJ/mol and −69 J/mol/K. The low value for DH# for the purified enzyme may indicate a simple heat induced chemical reaction, like release of an essential metal, for inactivation of the enzyme. A negative value for DS# for the purified SOD may indicate that protein unfolding was not important for inactivation and that aggregation of the molecules may have occurred16. The higher values for DH# and DS# for SOD activity in the crude extract indicate the presence of more thermostable isoenzymes.

Purification of SOD-C isoenzymes A purification scheme was devised primarily in order to optimise purification of the SOD-C isoenzymes from an aqueous extract of the wheat cultivar, Tonic. The total SOD activity and protein contents are given for each step and the recoveries (%) from each preceding step are presented in brackets (Table I). Preliminary experiments had indicated that the addition of Triton X-100 to the phosphate EDTA buffer resulted in extraction of more SOD activity and total protein. Precipitation with the 40–70% of saturated ammonium sulphate resulted in enrichment in SOD activity relative to total protein and with a purification factor of 4.7. After dialysis against 0·02  Tris-HCl buffer, pH 7·2, and adsorption to DEAE-Sephadex, fol-

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5.0 4.5 4.0 ln activity

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0

Figure 2 70°C.

5

10 15 20 Heating time (min)

5.0

–1

ln k (s )

5.5 6.0 6.5 7.0 7.5 8.0 2.95

3

3.05 3

3.1

3.15

3.2

–1

10 /T (K )

Figure 4 Arrhenius plots for heat inactivation. Α=SOD isoenzymes in crude wheat extract; Χ=purified SOD isoenyzme C.

5.0 4.5 4.0 ln activity

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0

30

Heat inactivation of superoxide dismutase activity in crude wheat extracts. Ε=50°C; Φ=55°C; Χ=60°C; Α=

4.5

8.5 2.9

25

5

10 15 20 Heating time (min)

25

30

Figure 3 Heat inactivation of the purified SOD-C isoenzyme. Φ=40°C; Ε=50°C; Χ=60°C; Α=70°C.

lowed by a single step elution with 0·4  NaCl, the specific activity was increased to 18·4, which was essential for further purification of SOD-C. Anion exchange chromatography with Q-Sepharose (Table II) resulted in the partial separation of the SOD isoenzyme groups A, B and C (Fig. 1) which were separated in the order B, C and A with increasing amounts of NaCl (Table II). The SOD-C isoenzyme fraction was purified further by preparative IEF and a purification factor of approximately 80 was achieved prior to chromatofocusing with a Mono-P column, when the main component was eluted as a sharp peak at around pH 5·7 (Fig. 5). Overall, after six purification steps followed by chromatofocusing, a 300fold enrichment of SOD activity in the form of SOD-C was achieved (Table I). The IEF gel shown in Figure 6 illustrates the level of purification achieved for the SOD-C type isoenzymes when stained for protein with silver. The finally purified SOD-C preparation, after preparative IEF, which effectively separated two SODC isoenzymes form the contaminating proteins, is shown in Figure 6. After chromatofocusing, the two electrophoretically similar SOD-C isoenzymes were eluted in 4 fractions and could not be fractionated further. The first fraction was considered to be the purest and was used for characterisation. The pIs of each SOD-C isoenzyme were determined by IEF to be 6·0 and 6·1. The Mrs of the SOD-C isoenzymes were estimated to be approximately 79 400 by PAGE, as shown by only one silver staining band. This indicated that the two closely separated SOD-C isoenzymes detected by isoelectric focusing, but

Wheat superoxide dismutase enzymes

Table 1 Purification stage

Purification of SOD-C from wheat

Bovine SOD equivalents (lg) (1)

Protein (mg) (2)

Ration (1)/(2)

13500 6970 (52) 4600 (34) 700 (15·2) 76 (21·5) 14·5 (29)

5580 612·0 (10·9) 250·0 (40·8) 20·0 (8) 0·04 (0·3) 0·02 (50)

2·4 11·3

1 4·7

18·4

7·7

Crude extract Ammonium sulphate precipitate Batch DEAE-sephadex Q-Sepharose Preparative IEF Chromatofocusing

Table 2 Purification by anion exchange chromatography on Q-Sepharose of wheat SOD isoenzymes Eluted fractions containing mainly SOD-B SOD-C SOD-A

NaCl Concentration () 0–0·15 0·15–0·2 0·2–1·0

SOD Protein Ratio (lg+) (mg) (1)/(2) (1) (2) 9000 750 400

20 11·3 15

45 66 26

∗ Bovine SOD equivalents.

not resolved by PAGE, were of similar size and possibly differed by only a few amino acids affecting the overall charge. No further attempt was made to separate the two SOD-C isoenzymes. The size of the subunits of SOD-C was determined in the presence of SDS and mercaptoethanol to be approximately 20 800, indicating that active SODC isoenzymes are mainly tetramers composed of similar subunits. Nevertheless, the two minor variants detected by PAGE may still reflect small differences in the structure of the subunits and Table III

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Purification factor

35·0

14

190·0

79

725·0

302

their assembly to form native tetrameric SOD-C. N-terminal amino acid sequence analysis showed that the N-terminal sequence of the SODC sequence tetrameric protein from the wheat cultivar, Tonic, was homologous with that of MnSOD from maize17. The residue identity between the Mn-SOD of maize and the wheat SOD-C was 57% and the number of identified residues that did not match accounted for only four out of 30 residues in the sequence (13%), with the remainder unidentified (X). Sequences within residues 32–61 for Mn-SODs from maize, human, yeast, E. coli and T. thermophilus also aligned with those of the wheat cultivar SOD-C isoenzyme. The boxed sequences shown in Table III are for homologous regions. The aligned sequences close to the N-terminal amino acid sequence of maize, human, yeast, E. coli and T. thermophilus were adapted from their complete sequences, which were presented in the aligned form by White and Scandalios17. The boxed sequences are regions of homology between all the amino terminal sequences. The N-terminal

N-terminal amino acid sequence analysis of wheat SOD-C and Mn-SODs from maize, human, yeast, E. coli and T. thermophilus

Source

Residue number 32

40 ∗ ∗ ∗ L X D L X Y D Y X A L P D L S Y D F G A L P D L P Y D Y G A

(1) Wheat (2) Maize (3) Human

X A T F T V T T V T K H S

(4) Yeast (5) E. coli (6) T. thermophilus

K V T L P S Y T L P P Y P F K L P

50

55

61

∗ L E X A V S X E I M X L X X Q L E P A I S G E I M R L H H Q L E P H I N A Q I M Q L H H S

D L K W D F G A L E P Y I S G Q I N E L H Y T S L P Y A Y D A L E P H F D K Q T M E I H H T D L G Y P Y E A L E P H I D A K T M E I H H Q

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100

Absorbance (280 nm, arbitrary units)

the SOD-C purified from wheat may have been an Fe-SOD or more likely was simply related to Fe-SODs, which is a characteristic of all MnSODs purified from other sources. In conclusion, the purified SOD-C from the wheat cultivar Tonic was found to contain two closely related tetrameric isoenzymes of Mr 80 000 and pI values of 6·0 and 6·1. N-terminal amino sequencing studies of SOD-C revealed extensive homology with Mn-SOD purified from maize. From the thermostability studies the low value of DH# for the purified SOD-C appears to indicate that enzymic inactivation occurs via a simple heatinduced chemical reaction, perhaps through the release of the bound metal. 1

5

10 15 Fraction number

20

Figure 5 Chromatofocusing on Mono-P of wheat SOD-C. Fractions of 0·5 ml were collected.

Acknowledgements We thank Dalgety plc for finncial support and the award of a studentship to S. M. Lawlor. We are pleased to acknowledge the amino acid sequencing carried out by Professor J. Findlay and his colleagues. REFERENCES

Figure 6 enzyme.

Isoelectric focusing of the purified SOD iso-

amino acid sequence of the purified wheat MnSOD contained invariant residues that have been conserved through evolution, indicating that the purified wheat SOD-C isoenzyme was probably a Mn-SOD. The residues marked ∗ are conserved throughout the amino terminal sequences of FeSOD from anaerobic, aerobic and photosynthetic bacteria18 and, therefore, it remains possible that

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Research’ (R.A. Greenwald ed.), CRC Press Inc., Boca Raton, Florida (1985) pp. 217–220. Asada, K., Takahashi, M. and Nagate, M. Assay and inhibitors of spinach superoxide dismutase. Agricultural and Biological Chemistry 38 (1974) 471–473. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (1970) 680–685. Haffner, P.H. and Coleman, J.E. Cu(II)—carbon bonding in cyanide complexes of copper enzymes. Journal of Biological Chemistry 248 (1973) 6626–6629. Asada, K., Yoshikawa, K., Takahashi, M., Maeda, Y. and Emmanji, K. Superoxide dismutases from a blue alga, Plectonera boryanum. Journal of Biological Chemistry 250 (1975) 2801–2807.

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15. Weisiger, R.A. and Fridovich, I. Superoxide dismutase organelle specificity. Journal of Biological Chemistry 248 (1973) 3582–3592. 16. Dannenberg, F. and Kessler, H.I.G. Reaction kinetics of the denaturation of whey proteins. Journal of Food Science 53 (1988) 258–263. 17. White, J.A. and Scandalios, J.G. Isolation and characterisation of a cDNA for mitochondrial manganese superoxide dismutase (SOD-3) of maize and its relation to other manganese superoxide dismutases. Biochimica et Biophysica Acta 951 (1988) 61–70. 18. Harris, J.I., Auffret, A.D., Northrop, F.D. and Walker, J.E. Structural comparisons of superoxide dismutases. European Journal of Biochemistry 106 (1980) 297–303.