PHENOLOXIDASE FROM THE LEAVES OF THE COTTON PLANT T. S. Yunusov and P. Kh. Yuldashev UDC 577.15.06 There is information in the literature on the presence of phenoloxldase in the leaves of the cotton plant [1]. We extracted the proteins from freshly frozen leaves with a tenfold amount of 0.1 M citrate-phosphate buffer, pH 7.2, in the presence of 1% of ascorbic acid and of Kapron powder - 0.5 g of powder per g of leaves. The extract was filtered and centrifuged at 18,000 rpm for 15 rain, and the supernatant liquid was clialyzedagainst distilled water for a day. The phenoloxidase activity in the dialyzate was determined by Drawert's method [2], using as the substrate mixture 2 ml of a 10% solution of catechol, 2 rrd of 2% prollne, and 1 rnl of enzyme solution. After the lapse of 20 rain, the reaction was fixed by the addition of 0.5 ml of a 5% solution of trichloroacetic acid. A change in the absorption (violet filter) by 0.001 in 1 rain was taken as the unit of activity. To determine the highest specific activity of the phenoloxidase in the leaves of the cotton plant, we studied the dynamics of the accumulation of this enzyme in young cotton shoots. The experiments were performed on cotton-plant leaves collected in the early periods of vegetation in the Tashkent .blast in 1972. No phenoloxidase was found in the seeds and the cotyledon.us leaflets. The specific activity of the phenoloxidase was 7 in May, 14 in June, 50 in July (chopping period), 40 in August, and 10 in September. Con- sequently, the greatest phenoloxidase activity is observed in the period of vigorous growth of the cotton plant. In addition to the soluble phenoloxidase, we found in the leaves of the cotton plant a latent phenoloxidase which was extracted by the buffer solution mentioned but only in the presence of the detergent Triton ~-100. All the operations were performed at a temperature of +4-6°C. Thus, the optimum conditions have been selected for the extraction of phenoloxidase from the leaves of the cotton plant of variety 108-F, and the vegetation period with the greatest specific activity of this enzyme has been determined. 1. 2. LITERATURE CITED E. E. King, Phytochem., 1._00,23370971). F. Drawert and H. Gebbing, Naturwiss., 50, 522 (1963). Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 446-447, May-June, 1973. Original article submitted January 5, 1973. © 19 75 Plenum Publishing Corporation, 227 West 17th Street, New York, N. Y. 10011. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microplming, recording or otherwise, without written permission of the publisher. A copy of this article is available from the publisher for $15.00. 429

Phenoloxidase from the leaves of the cotton plant

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P H E N O L O X I D A S E F R O M T H E L E A V E S O F T H E C O T T O N P L A N T

T . S . Y u n u s o v a n d P . K h . Y u l d a s h e v UDC 577.15.06

There is informat ion in the l i t e r a tu r e on the p re sence of phenoloxldase in the l eaves of the cotton plant [1].

We ex t rac ted the p ro te ins f r o m f re sh ly f rozen l eaves with a tenfold amount of 0.1 M c i t ra te -phospha te buffer , pH 7.2, in the p re sence of 1% of a sco rb ic acid and of Kapron powder - 0.5 g of powder p e r g of l eaves . The ex t r ac t was f i l te red and centr i fuged at 18,000 r p m for 15 rain, and the supernatant liquid was cl ia lyzedagainst dist i l led wa te r for a day. The phenoloxidase act ivi ty in the dialyzate was de te rmined by D r a w e r t ' s method [2], using as the subs t ra t e mix ture 2 ml of a 10% solution of catechol , 2 rrd of 2% prol lne, and 1 rnl of enzyme solution. After the lapse of 20 rain, the reac t ion was fixed by the addition of 0.5 ml of a 5% solution of t r i ch lo roace t i c acid. A change in the absorpt ion (violet f i l ter) by 0.001 in 1 rain was taken as the unit of act iv i ty .

To de te rmine the highest specif ic act iv i ty of the phenoloxidase in the leaves of the cotton plant, we studied the dynamics of the accumula t ion of this enzyme in young cotton shoots . The expe r imen t s were p e r f o r m e d on cot ton-plant l eaves col lected in the e a r l y per iods of vegetat ion in the Tashkent . b l a s t in 1972. No phenoloxidase was found in the seeds and the co ty ledon.us l ea f le t s . The specif ic act ivi ty of the phenoloxidase was 7 in May, 14 in June, 50 in July (chopping period), 40 in August, and 10 in September . Con- sequently, the g r e a t e s t phenoloxidase act ivi ty is obse rved in the per iod of vigorous growth of the cotton plant.

In addition to the soluble phenoloxidase, we found in the leaves of the cotton plant a la tent phenoloxidase which was ex t rac ted by the buf fer solution mentioned but only in the p re sence of the detergent Tr i ton ~-100 . All the opera t ions were p e r f o r m e d at a t e m p e r a t u r e of +4-6°C.

Thus, the op t imum conditions have been se lec ted for the ex t rac t ion of phenoloxidase f r o m the leaves of the cotton plant of va r i e ty 108-F, and the vegetat ion per iod with the g r e a t e s t specif ic act ivi ty of this enzyme has been de te rmined .

1. 2.

L I T E R A T U R E C I T E D

E. E. King, Phytochem., 1._00, 23370971) . F. Drawer t and H. Gebbing, Naturwiss . , 50, 522 (1963).

Insti tute of the C h e m i s t r y of Plant Substances, Academy of Sciences of the Uzbek SSR. Trans la ted f r o m Khimiya Pr i rodnykh Soedinenii, No. 3, pp. 446-447, May-June , 1973. Original a r t i c le submit ted January 5, 1973.

© 19 75 Plenum Publishing Corporation, 227 West 17th Street, New York, N. Y. 10011. No part o f this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microplming, recording or otherwise, without written permission o f the publisher. A copy o f this article is available from the publisher for $15.00.

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