L.) (ORYZA SATIVA

Indian J. Plant PIIYliol., Vol. XXVII, No.3 pp. 271-275 (September 1984)

SUBSTRATE SPECIFICITY OF PEROXlDASE ISOZYMES IN RICE

(ORYZA SATIVA L.)

V. DANGE AND G.M. REDDY

Department of Genetics, Osmania University, Hyderabad-500 007

SUMMARY

The substrate specificity of peroxidase isozymes of ten day old rice seedlings was studied following ammonium sulphate fractionation and further seperated using disc gel electrophoresis. There was a considerable variation in the total number of isozyme bands when a few aromatic amines and phenolic compounds were used as hydrogen donors. A greater number of iso'l.ymes were stained with benzidine, guaiacol and O-pheny­ lenediamine, while a

phloroglucinol.

lesser number was observed with P-toluidine and

INTRODUCTION

Peroxidase catalyses a two step oxidation of various substrates in the presence of hydrogen peroxide and the enzyme kinetics can be easily studied by spectroscopy and also electron transfer processes (Dunford and Stillman, ]976).

Peroxidase isozymes were first detected by Thoren, (1942) in horse radish roots, although these were present throughout the plant kingdom (Shin and Nakamura, 1962). These isozymes appear to catalyze the same reaction, how­ ever individual isozymes may differ with respect to their physico-chemical properties. molecular weight. pH and temperature optima. also kinetic pro­ perties like Km and Vmax and substrate specificities. The multiple molecular forms of peroxidase isozymes show difference regarding their preference for diverse hydrogen donors (Farkas and Stahrman. 1966; Markund et al., 1974).

The present study mainly deals with the specificity of anionic peroxidase isozymes of rice seedlings to diverse hydrogen donors which are either phenolic compounds or aromatic amines.

MATERIALS AND METHODS

•

•

272 V. DANGE AND G.M. RIIDDY

Ammonium sulphate fraction : The filtrate was brought to 35% ammonium sUlphate saturation and centrifuged at 10,000 rpm for 15 minutes. The pellet was discarded and the supernatant was brought to 90% ammonium sulphate saturation. After standing overnight, the solution was centrifuged at 10,000 rpm for 15 minutes. The pellet was dissolved in 0.005 M Tris-HCL buffer (pH 7.0) and then dialyzed against 0.005 M Tris-HCl (pH 8.0) containing 0.1 M KCLT for 48 hours. The dialysate was centrifuged at 10,000 rpm for 15 minutes and the supernatant was lyophilised and designated as crude peroxidase fraction.

Polyacrylamide gel electrophoresis (PAGE): The anionic peroxidase isozymes were separated by disc PAGE by the method of Davis (1964), the staining mixture contained 0.1 M of phenolic compounds (guaiacol, pyrogallol, phloro­ glucinol, catechol and O-amino-phenol), (0.1

%

inum Chloride per 100 mI, and the isoperoxidases were also stained with 0·05 M aromatic amines as hydrogen donors (benzidine, O-diansidine, O-phenylenedia­ mine and P-toluidine). The conditions for resolution and staining for isozymes, the amounts of protein applied andlthe substrate concentrations were chosen after preliminary work with a view to stain the possible number of isozymes. The stained peroxidase isozymes pattern was scanned with Shimadzu UV-240 Spectrophotometer.

RESULTS

Eight isoperoxidases were stained with guaiacol, O-aminophenol, pyrogallol and catechol. However, with phloroglucinol as a substrate only three isozymes were stained. These corresponded with one, two and eight isozymes (Fig. 1). Comparison of the isozyme-scanning pattern oDtained with

2

A c D _{• E} A c

...

5

I •

L -_ _ _ _- L_ _ _ _ _ _L -_ _ _ _~_ _ _ _~_ _ _ _ ~~ _ _ _ _ ~ _ _ _ _ ~ _ _ _ _ ~ _ _ _ _~

...- - - ' , - MIGRATION " - - - ' , -MIGRATION­

+ o + o

Fig. 1. Scanse of the anionic peroxidase isozymes, stained with phenolic compounds as hydrogen donors; A-guaiacol; B-catechol; C-pyrogallo); D-O-aminophcnol; E-phloroglucinol.

SEED SETTING IN SUNFLOWER 273 guaiacol, pyrogallol. catechol and O-aminophenol, revealed that isozymes one and eleven did not stain with O-aminophenol. but stained for three and nine isozymes. These two isozymes were not observed with guaiacol, catechol and pyrogaJlol. The intensity of staining individual isozymes with O-aminophenol, catechol, guaiacol was fonnd to he better than that observed with pyrogallol. In scans of the anionic peroxidase isozymes stained with aromatic amines as hydrogen donors, thirteen isozymes were noticed wiili benzidine (Fig. 2), eleven with O-phenylenediamine and nine with O-dianisidine and the least of three were stained with P-toluidine. The differences in the total number and the relative activity of the individual isozymes differed markedly with different

Table-I Rm values of the anionic iso zymes of peroxidase with phenolic compounds as hydrogen donors

OH

hor,,,,.. &

'....:

,

..

"

11 0-0"2 0·071 0185 0-257 01"0

0 .. 80

0660 0710 07..0 OCH 3 OH

& O H

I"'"_....: - ColtOO! 0·011 .. 0-071 0220 0·108 0501 0-671 0722 0770 P,ro,oHol 0·0 ..2 0·071 0-190 0260 0142

0· ..71

0·710

0·7.. 0

OH

&NHZ I....:

O-Amlnol Phenol

0·071 0·128 0·270

0357

0 .... 2

0·557

'0·6811

0·71 ..

OH

HO

~

PIIloro ,'''CIIlOL

0·0117

0·0'..

0·5118

hydrogen donors. A comparison of isozyme scans obtained with aromatic amines viz., benzidine. O-dianisidine and O-phenylenediamine, revealed that the isozymes of six and ten did not stain with O-phenylenediamine; On the other hand isozymes three, seven, twelve and thirteen did not stain with O-diani­ sidine. Thus it is evident that the isozyme moving more towards the anode were more effectively stained with benzidine and O-pheylenediamine.

DISCUSSION

274

~~'1111

r -" '

V. DANGE AND G.M. REDDY

Table-2 Rm values of the anionic isozymes of peroxidase with aromatic amines as hydrogen donors.

O N H 2 loozym. . _{H2 N}

--V--V

NH2

Q

~I

OCH3 OCH3 _NH2

BfnZldine 0- d,anlidlne 0- Phenylene P-Ioluidi ••

diamine

0·061 0·061 0·057 0·055

2 0 128 o 114 0-114 0-080 3 0 178 0·142

4 o 250 0·180 0- 200 5 0 290 0265 O· 265 6 0 342 0·324

7 o 430 6·371

8 0 481 o 438 o 500 o· 520 9 0·557 o 585 0-610

10 0 490 o 628

II 0 657 0-685 0-700 12 0·714 0·710

13 o 757 0·770

peroxidase isozymes depends on the nature of the hydrogen donor (Macko and Novacay. 1966). although substrate specificity varies considerably among the individual isozymes (Evans and Allridge, 1965). In the present investigation, the total number of isozymes as weH as relative activity assessed by Rm values were found to be different. The isozymes 12 and 13 moving fast towards the anode. oxidised all the phenolic substrates more effectively. Isozymes-3 and 9 were stained with O-aminophenol but not with guaiacol, catechol and pyrogallol, where as isozyme-l was stained with these three compounds. O-aminophenol was found ineffective towards least migrated isozymes. Among the phenolic compounds, catechol and guaiacol were effective in staining isozyme-ll but not pyrogallol and O-aminophenol. Thus the anionic peroxidase isozymes manifested more intensive substrate in perference to bivalent phenols (guaiacol. catechol) than trivalent ones (Phloroglucinol, O-aminophenol).

275

PEROXIDASE lSOZYMBS IN RICE

co.mpo.unds like Benzidine and guaiaco.l play an impo.rtant ro.le as hydro.gen do.no.rs.

The iso.zymes mo.ving to.wards the ano.de were actively stained with benzidine. O-phenylenediamine. guaico.l and catecho.l. The slo.w Iy mo.ving iso.zymes were fo.und active, when stained with benzidine and O-phenylenedi­ amine and less active, co.mpared to. phlo.ro.glucino.l. P-to.luidine, benzidine and O-phenylenediamine amo.ng the aro.matic amines were mo.re effective hydro.gen do.no.rs fo.r staining not only fast mo.ving iso.zymes but also. slow moving ones as co.mpared to o.ther substrates.

Biolo.gical {unctions o.f pero.xidases may be related to substrate specificity. In o.therwo.rds, the availability o.f numero.us substrates for mUltiple peroxidase iso.zymes, with many diverse metabo.lic functio.ns may be attributed to. per­ o.xidase isozymes in higher plants which might have played an important role in the evo.lution of species.

ACKNOWLEDGEMENTS

The autho.r gratefuJly acknowledge the financial assistance pro.vided by Indian Co.uncil of Agrl. Research (ICAR) Krishi Bhavan. New Delhi, and partial financial Assistance by USDA, Grant No.. FG/2n-585 (In-SEA-157) New Delhi.

REFERENCES

Davies, B.J. (1964). Disc electrophoresis of Methods and application to human serum proteins. Annl. N.Y. ACQd Sci., 121 : 404.

Dunford, H.B. and Stillman, J.S. (1976). On the function and mechanism of action of peroxi­ dase. Co-ordination Chemistry, Review., 19 : 187-251.

Evans, J.J. and Allridge, N.A. (1965). The distribution of peroxidases in extreme dwarf and normal tamato, (Lycopersicon esculentum : Moo). Phytochemistry, 4 : 499-503. ,Farkas, G.L. and Stahman, M.A. (1966). On the nature of changes in peroxidase isoenzymes

in bean leaves infected by Southern Bean Mosaic Virus. Phytopathology, 56 : 669-677.

Macko, V. and Novacky, A. (J966). Contribution to the study of plant peroxidase isozymes by means of disc electrophoresis on acrylamide gel. Biologis (Bratislava) 21 : 128-133.

Marklund, S., Ohlsson, P.L, Opara, A., and Paul. K.G. (1974). The substrate. profiles of the acidic and slightly basic horse radish peroxidases. Biochem. Biophysic. Acta., 350 : 304-313.

Shin, M. and Nakamura. W. (1962). Indole acetic acid oxidase activity of wheat peroxidase. J. Biochemi.• 52: 444-451.

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