420 J. Inst. Brew.. November-December. 1983, Vol. 89, pp. 420-423

SUPEROXIDE DISMUTASE IN BARLEY

ByC. W. Bamforth*

(Brewing Research Foundation, Nutfield, Surrey)

Received 23 March 1983

Superoxide dismutase occurs in barley where it can be measured using a xanthine oxidase assay,but not using an assay based on the reduction of Nitro Blue Tetrazolium by photo-reducedriboflavin. Three isozymes are present in the grain: two are cyanide-sensitive and one, of loweractivity, is not. Superoxide dismutase is mainly in the embryo of barley and develops in theacrospire, rootlets and endosperm during germination. Synthesis of this enzyme is acceleratedby exogenous gibberellic acid. Significant quantities of superoxide dismutase survive kilning:lager malts contain ca 50% more than do ale malts. The enzyme is rapidly destroyed duringmashing at 65°C, but large amounts survive mashing at 45°C.

Key words: analysis method, enzyme, mailing, mashing,

oxidation.

Introduction

The superoxide radical, O2" (or HOj-, pKa = 4-88) is

generated from oxygen in numerous autoxidation and

enzyme-catalysed reactions.10 Very small quantities ofsuper-

oxide, or possibly singlet oxygen15 and the hydroxyl radical,

which are derived from it,7 can have a number of damaging

effects on biochemical systems. Those effects which would

seem to be or importance in a brewing context arc the

peroxidation of lipids (and, through this, the cross-linking

or proteins), the degradation of polysaccharides and the

inactivation of enzymes.8-912

To prevent the accumulation of superoxide, aerobic

organisms have developed the enzyme superoxide dismu

tase,9 which catalyses the reaction

O2- 2H+ O2

Most inferences concerning the harmful effects of super-

oxide have been drawn from studies of the protective action

of the enzyme. For example, a patent exists on the use

ofsuperoxidc dismutase to prevent the browning of apples,

the staling of potatoes, carrots and mushrooms and the

inactivation of enzymes.7

In view ofthe probable contribution ofsuperoxide in caus

ing the oxidation of lipids,11" it can be anticipated that

superoxide dismutasc in barley and malt will prevent the

development of rancid notes during grain storage and of off-

flavours in beer. Superoxide dismutase activity has been

demonstrated in barley seeds,16 but there is no record of how

many such enzymes occur in the grain, nor of what their

properties are in relation to malting and brewing. The

present paper rectifies this situation.

305 mm w/s paper. Worts were maintained at 4°C for 18 h

before they were assayed for superoxide dismutase.

Extraction ofgrain.—This was carried out as described by

Bamforth el aP except that SO mM potassium phosphate, pH

7-8 was used and the extraction period was 1 h. Isolated tis

sues were ground in a mortar and pestle prior to the addition

of buffer.

Assay ofsuperoxide dismutase.—

(i) Xanthine oxidase-cytochrome c assay. Disposable

1 cm-light path plastic cuvettes were used containing

150 umol of potassium phosphate, pH 7-8; 0-3 umol EDTA;

003umol ferricytochrome c; 0-15umol xanthine; sufficient

xanthine oxidase to cause an increase in absorbance at

550nm of 001-0025/min; enzyme and water to a total

volume of 3 ml. The mixtures were incubated at 25°C for 2

min without xanthine, which was then added to start the

reaction. The reduction of cytochrome c was followed by

recording the increase in A5SO over a period of 3 min.

(ii) Nilro Blue Tetrazolium (NBT) assay. Disposable

1 cm-light path plastic cuvettes were used containing

150 umol of potassium phosphate, pH 7-8; 0-007 umol of

riboflavin; 30 umol of mclhioninc; 0-17 umol of NBT;

enzyme and water to a total volume of 3 ml. The reaction

was started by adding riboflavin and placing each cuvette

3-5 cm from a 15W fluorescent light within an aluminium

box. After 6 min, the cuvettes were removed to a dark con

tainer until absorbances at 560 nm could be read.

Polyacrylamide gel electrophoresis.—This was performed

in 7% polyacrylamide gels at pH 8-3 by the method of

Davis.6 Superoxide dismutasc activity was located by the

method described by Beauchamp & Fridovich.4

Experimental

Barley.—Unless stated otherwise, all the experiments were

performed with Maris Otter. Dissection of the embryo and

scutellum and the isolation of the aleurone and endosperm

were performed as described previously.1 The malting and

storage of green malt samples was as described elsewhere.2

Gibberellic acid (0-125 ppm) was added as a spray at casting.

Mashing.—An ale malt (Proctor) was milled at a setting

of 0-7 mm on a Miag Disc Mill and 50 g portions weremashed at a liquor: grist ratio of3 : 1 using the BRF mashing

bath. After 15,30,45 or 60 min stirring at different temperatures, mashes were brought to 450 g with deionised water at20°C and (after a 30 min stand) were filtered through Postlip

'Present Address: Group Research Laboratory, Bass Brewery Ltd,

137 High Street, Burton-on-Trcnt. DEN 1JZ.

Results

Assay.—Assay (i) is routinely used for the measurement

of superoxide dismutasc in barley (see later). The methodrelies on the inhibition by superoxide dismutase of the

reduction of cytochrome c by supcroxide produced in the

oxidation of xanthine.

xanthine

uric acid

cytochrome c (Fe2+)

cytochrome c (Fe1*)

supcroxide

dismutase

Vol. 89, 1983] bamforth: supi-roxide dismutase 421

08 1004 00

Protein fmg)

Fig. I. Influence of protein concentration on % inhibition of

cytochromc c reduction.

Using this assay, the reduction ofcytochrome c is inhibited

in proportion to protein concentration in crude extracts of

barley up to ca 80% inhibition (750 ug protein) (Fig. I). One

unit ofsuperoxidc dismutase is defined as the amount which

lessens the rate of reduction of cytochrome c by 50%.

Levels ofsuperoxide dismutase in barley and development

during malting.—Different varieties of barley grown at a

single site contained levels ofsuperoxide dismutase between

1-2 and 2-9 units/corn (Table I). There was no apparent

relationship between levels of the enzyme and the malting

grade ofthe barley. The variety Triumph, grown at different

locations in the UK, contained activities of superoxidc

dismutase between 2-2 and 3-0 units/corn (Table II).

Superoxidc dismutase increases very slightly in amount

during steeping, but is present in green malt at levels over

5-fold higher than in barley (Fig. 2). This enzyme develops

TABLE I. Superoxide Dismutase in

Different Barley Varieties

Variety

Goldmarkcr

Atcm

KymTriumph

Flare

KoruGcorgic

Ark Royal

Units/corn

1-252002102-242-402-40

2-74

2-91

sooner when barley is germinated in the presence of exo

genous gibberellic acid (GA), although similar quantities of

superoxidc dismutase are present in green malt after four

days ofgermination in the presence or absence ofadded GA.

After drying green malt for 24 h at 45°, 65°, 85° and IO5°C

respectively, the survival of the activity was 81, 52, 18 and

0%.

Location of superoxide dismutase in barley and green

malt.—The embryo plus scutellum from barley contains

about 2-1 units ofsuperoxide dismutase per corn, whilst the

aleurone contains about 0-3 units/corn. The starchy endo

sperm contains no detectable level. Both the acrospire and

rootlets of germinated grain contain about 3-0 units/corn.

Superoxide dismutase in various cereal preparations.—

Dried samples of experimental malts produced with and

without added GA each contained approximately 3-5 times

more superoxide dismutase than did the original barley

(Table III). Commercial lager malts contained rather more

of the enzyme than did the ale malts. Flaked or torrefied

adjuncts contained very little superoxide dismutase, which

was present, however, in wheat flour. Especially large

amounts of the enzyme were found in soya beans, even after

heating in a microwave oven (250 g beans; Tappan oven

Model 56-0478, full power for 5 min).

Fig. 2. Development of superoxide dismutase during malting. (O)Green malt produced with added GA; (•) Green malt producedwithout added GA. Steeping and germination were at 16°C. Thebarley variety was Sonja.

TABLE II. Superoxide Dismutasc inTriumph Grown at Different Sites

Site

AB

C

DEFG

Units/corn

2-892-24

2-702-64

2-97

2-782-44

Effect of pH on superoxide dismutase in barley.—Determination of the precise pH optimum for superoxide

dismutasc is rendered difficult by the pH dependence of the

xanthinc oxidase reaction. As xanthine oxidase acts ca

5 x 104 times more rapidly at pH 7-8 as compared to pH 4-5,

then even when the amount ofxanthine oxidase added to the

assay is greatly increased, only low AA,50 values are

measured, preventing the accurate measurement of %

inhibition by supcroxide dismutase. Nevertheless, it was

apparent that significant dismutation occurs at pH values aslow as 4-3, showing that the enzyme could act at wort or beer

pH's.

422 hamforth: supkroxide dismutase [J. Inst. Brew.

TABLE HI. Supcroxide Dismutase in Cereal Preparations

Cereal

Sonja barleySonja malt, produced with CASonja malt, no GA

Commercial ale malts AB

CCommercial lager malts I

11

IIIFlaked barley

Torrefied barleyWheat flourTorrefied wheat

Flaked maizeRaked rice

Soya beans

Micronised soya beans

Superoxide dismutasc(units/lOg)

4701600

1600960

11208001600

17601420

00

56080

4343

6400

2400

Effect ofheat on superoxide dismutase in barley.—When

a crude extract of barley was heated at 59°C for 20 min

approximately 70% of the superoxide dismutasc was de

stroyed. Upon heating at 80°C or 100°C, activity was

completely destroyed within 20 min.

Activity ofsuperoxide dismutase during mashing.—Most

ofthe superoxide dismutase in malt was destroyed within 15

min of mashing at 65°C, and destruction was complete with

in 30 min (Fig. 3). However, large amounts of activity

survived a one-hour mash at 45°C, and significant amounts

of superoxide dismutase were present after 30 min of mash

ing at 55°C.

30

Tims of finishing (mini

45 60

Fig. 3. Supcroxide dismutase in worts after different periods of

mashing at various temperatures. The malt contained 56 units

of supcroxidc dismutase/g. (O) 45°C; (•) 55°C; (A) 65°C; (A)

75°C.

Multiplicity of superoxide dismutases in barley.—Three

bands ofsuperoxide dismutase were separated by polyacryl-

amide gel elcctrophoresis of extracts of barley. The values of

RF (relative to Bromophenol Blue) were 0-20,0-64 and 0-81.

The slowest migrating band appeared only after overnight

incubation of the gels in the assay solution, indicating it to

be either of low catalytic activity or present in very small

quantity. Neither of the faster moving bands appeared if

200 um potassium cyanide was included in the assay mix

ture, whereas the third band did so. In view of the known

response of different superoxide dismutases to the presence

of cyanide, it seems that the faster migrating bands are

typical of the copper-and zinc-containing enzymes found

in eukaryotes whilst the slowest moving protein is prob

ably a manganese containing supcroxide dismutase of

mitochondrial origin.9

Problems with using the NBT assayfor barley superoxide

dismutase.—The NBT assay relies on the inhibition by

superoxide dismutase of the reduction of Nitro Blue Tetra-

zolium by photo-reduced riboflavin, following the scheme:

Riboflavin

hu

Reduced Riboflavin

superoxide dismutasc

Nitro Blue

Tetrazolium

Formazan

Using this assay it was found that up to 60% ofthe enzyme

in extracts of barley apparently survived boiling for 30 min,even though no bands of superoxide dismutasc could be

located when boiled extracts were examined by poly-

acrylamide gel clcclrophoresis. This 'apparent superoxide

dismutase' was present in extracts of barley malt as well as

in beer and it was non-dialysable. It seems unlikely to be a

result ofthe channelling away ofsuperoxide radicals as it was

not detected using the xanthine oxidasc-cytochromc c assay.

Its occurrence renders the NBT assay unacceptable for the

measurement of superoxide dismutase in barley and malt.

Discussion and conclusions

It is probable that supcroxide dismutase is present in all

aerobic organisms.12 In barley, the enzyme is mostly located

in the embryo, with smaller amounts in the aleurone. By

contrast, the non-respiring starchy endosperm contains no

detectable superoxidc dismutase. This is at variance with the

findings of Giannopolitis & Ries,11 who found that 62% of

the enzyme in the seeds of oats was located in the endosperm. In view of the vigorous gaseous exchange which

occurs in the embryo and, to a lesser extent, in the aleurone

during germination, with the accompanying greater gener

ation of superoxide, it is perhaps not surprising that these

tissues contain the largest amounts of protective superoxide

dismutase. The present finding that this enzyme is as pre

valent in the acrospire as in the rootlets ofgerminated barley

also differs from the observation that three times as much

activity is associated with the shoots as compared to the roots

in oats, peas and maize."

Superoxide dismutase in barley and green malt is com

paratively heat stable, but is rapidly destroyed in boiling.

Again this does not agree with the findings of Giannopolitis

& Ries,11 who concluded that 100,30 and 20% ofthe super

oxide dismutase activity in maize, oats and peas respectively

will survive boiling for 20 min, oddly citing its sensitivity to

cyanide and its non-dialysability as evidence for enzymic

activity. They made no attempt to locate such activity bystaining gels. Giannopolitis & Ries employed the NBT assay,

which has now been shown to be unsuitable for measuring

superoxide dismutase in barley and it remains to be ascer-

Vol. 89, 1983] BAMTORTH: SUPEROXIDI: mSMUTASE 423

tained whether similar, non-enzymic factors arc responsible

for residual 'activity' in maize, oats and peas. Nevertheless,

using the xanthinc oxidase-cytochromc c assay, it has now

been shown that much supcroxide dismutasc in soya beans

is stable to rigorous cooking, which destroys lipoxidase.'

The pattern of barley supcroxide dismutases fractioncd on

polyacrylamide gels is reminiscent of that found for wheat

grain,' viz two fast moving enzymes which are inhibited by

cyanide and a slow moving cyanide-insensitive enzyme,

suggesting that very similar enzymes are involved.

Although unproven, it is assumed that compounds re

sponsible for the stale flavours which can arise during storageof beer are the degradation products of oxidised derivatives

of unsaturated fatty acids. However, the stage(s) in the con

version from barley to beer when this oxidation takes place

are uncertain, and proof, one way or the other, must await

the development of ready methods for measuring the

oxidised derivatives. It is most likely that non-enzymic

oxidation by superoxide will occur throughout the process

and in the finished product, and thus the presence of super-

oxide dismutasc at all stages would be desirable. The use of

lager-type kilning regimes allied to low temperatures for

the commencement of mashing will favour supcroxide

dismutation. However at higher temperatures, supcroxide

dismutasc will be destroyed, whilst the supcroxide radical

could still be produced. The use of heat-stable dismutases

would then be desirable, soya beans providing one such

source.

No supcroxide dismutasc will survive into beer. It remains

to be ascertained whether its addition will improve the

flavour stability of beer, and whether it will serve as a prefer

able alternative to glucose oxidase as a method for improving

shelf-life.

Acknowledgements.—This paper is published with the

kind permission of the Director of the Brewing Research

Foundation. Mr C. D. Meller and Mrs H. L. Martin are

thanked for excellent assistance with the experimentation.

REFERENCES

1. Bam forth, C W. & Martin, H. L., Journal of the Institute of

Brewing. 1981,87, 81.2. Bamforth, C. W., Martin, H. L & Wainwrighi. T.. Journal of

the Institute ofBrewing. 1979, 85, 334.3. Baxter, E. D., private communication.4. Bcauchamp, C. O. & Fridovich, 1., Analytical Biochemistry.

1971,44,276.

5. Bcauchamp, C. O. & Fridovich, I., Biochimica et BiophysicaActa. 1973,317,50.

6. Davis, B. J., Annals of the New York Academy of Sciences.1954,121,404.

7. Elstner, E. F., Annual Reviews of Plant Physiology. 1982, 33,

8. Eriksson, C. E., Food Chemistry. 1982,9,3.9. Fridovich, I., Annual Reviews of Biochemistry. 1975,44, 147.10. Fridovich, I., Science. 1978, 201, 875.11. Giannopolitis. C. N. & Rics, S. K... Plant Physiology. 1977.59,

309.

12. Halliwell, B., Trends in Biochemical Sciences. 1982, 7, 270.13. Kellogg, E. W. & Fridovich, 1., Journal ofBiological Chemistry.

1975.250,8812.14. Kcllogg, E. W. & Fridovich, 1., Journal ofBiological Chemistry.

1977,252,6721.15. Krinsky, N. I.. Trends in Biochemical Sciences. 1977. 2. 35.16. Matkovics, B., Superoxide and Superoxide Dismutases. Eds:

A. M. Michclson, J. M. McCord & I. Fridovich, AcademicPress, 1977,501.

17. Michelson, A. M. & Monod, J., United States Patent No. S 920521.