SOME PROPERTIES OF THE PHENOLOXIDASE OF THE COTTON PLANT T. S. Yunusov and P. Kh. Yuldashev UDC 577.15.06 We have investigated the nature and some properties of the phenoloxidase that we isolated from the cotton plant [1, 2]. The content of copper in the phenoloxidase has been studied. This was determined spec- trophotometrically at 440 nm [3] from a calibration curve. The protein was dissolved in distilled water, distilled in a glass apparatus with a quartz condenser, and dialyzed against the same water for two days. Then the protein solution was freeze-dried, and the copper was determined. The protein was digested in quartz test-tubes at 120°C for 3 h, a weighed sample being dissolved in 1 ml of concentrated H2SO 4 (KhCh [nchemically pure"]) and one drop of HNO3 (KhCh) (the results are summarized in Table 1). The results of an investigation of the dependence of the activity of the phenolaxidase on the pH showed that the enzyme has a pH optimum between 7.6 and 7.8. At this value of the pH there are two temperature optima- at 30 and 60°C , the activity at 60°Cbeing2.5 times greater than that at 30°C. In view of the fact that the enzyme exhibited a high activity at elevated temperatures, we studied the stability of the phenoloxidase at 70°C (see Fig. 1). As can be seen from the graph, the enzyme is completely inactivated in 30 min. A 2 10 ?0 3O Time, min // , , i * , i * * ° l o, oe o,o~ u.t~ tlt~ 0,e- mg/ml Fig. 1 Fig. 2 Fig. 1. Stability of the enzyme at 70°C. Fig. 2. Dependence of the activity of the phenoloxidase on the concentration of the actual enzyme. TABLE 1 Expt. Sample, mg I } 1,5 I1 I !1 } 2 I No. of Fg of eMean value CuS+inImg per 1 mg of protein of protem 10 8,3 9,1Fg 9,3 8,7 Cu 2+ in the protei n, % 0.9 Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 541-542, July-August, 1974. Original article submitted February 26, 1973. ©19 76 Plenum Publishing Corporation, 22 7 West 17th Street, New York, N. Y. 10011. No part of this publicatioti may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without ~vritten permission of the publisher. A copy of this article is available from the publisher for $15.00. 561

Some properties of the phenoloxidase of the cotton plant

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

OF 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 . Kh . Y u l d a s h e v UDC 577.15.06

We have investigated the nature and some proper t ies of the phenoloxidase that we isolated f rom the cotton plant [1, 2]. The content of copper in the phenoloxidase has been studied. This was determined spec- t rophotometr ica l ly at 440 nm [3] f rom a cal ibrat ion curve. The protein was dissolved in distilled water, dist i l led in a glass apparatus with a quartz condenser, and dialyzed against the s a m e water for two days. Then the protein solution was f reeze-dr ied , and the copper was determined. The protein was digested in quartz tes t - tubes at 120°C for 3 h, a weighed sample being dissolved in 1 ml of concentrated H2SO 4 (KhCh [nchemically pure"]) and one drop of HNO 3 (KhCh) (the resul ts a re summar ized in Table 1). The resul ts of an investigation of the dependence of the activity of the phenolaxidase on the pH showed that the enzyme has a pH optimum between 7.6 and 7.8. At this value of the pH there a re two tempera ture o p t i m a - at 30 and 60°C , the activi ty at 60°Cbeing2.5 t imes g rea t e r than that at 30°C. In view of the fact that the enzyme exhibited a high activity at elevated temperatures , we studied the stability of the phenoloxidase at 70°C (see Fig. 1). As can be seen f rom the graph, the enzyme is completely inactivated in 30 min.

10 ?0 3O Time, min

// , , i * , i * * ° l

o, oe o,o~ u . t~ tlt~ 0,e- mg/ml

Fig. 1 Fig. 2

Fig. 1. Stability of the enzyme at 70°C.

Fig. 2. Dependence of the activity of the phenoloxidase on the concentration of the actual enzyme.

TABLE 1

Expt. Sample, mg

I } 1,5 I1

I !1 } 2

No. of Fg of e Mean value CuS+inImg per 1 mg of protein of protem

10 8,3 9,1Fg

9,3 8,7

Cu 2+ in the protei n, %

0.9

Institute of the Chemis t ry of Plant Substances, Academy of Sciences of the Uzbek SSR. Transla ted f rom Khimiya Pr i rodnykh Soedinenii, No. 4, pp. 541-542, July-August, 1974. Original ar t ic le submitted F e b r u a r y 26, 1973.

©19 76 Plenum Publishing Corporation, 22 7 West 17th Street, New York, N. Y. 10011. No part o f this publicatioti may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without ~vritten permission o f the publisher. A copy o f this article is available from the publisher for $15.00.

561

Page 2: Some properties of the phenoloxidase of the cotton plant

We have also investigated the dependence of the activity of the phenoloxidase on its concentration; this re la t ionship is l inear (Fig. 2).

lo 2. 3.

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

T. S. Yunusov and P. Kh. Yuldashev, Khim. Pr i rodn . Soedin., 446 (1973). T. S. Yunusov and P. Kh. Yuldashev, Khim. Pr i rodn . Soedin., 122 0-974). S. C. Dhar and S. M. Bose, Leather Science, 1_~2, 54 0-965).

562