3 4 0 Abstracts

F051 CHARACrERIZATION AND REAC'YIVITY OF THE PRINCIPAL ANIONIC AND CATIONIC ISOENZYMES OF "ZUCCHINI"

P E R O X I D A S E S b c c c

A . M a r c h e s l n i a, ~ , L . C a s e l l a , M . G u l l o t t i , E . L a u r e n t l and S .Pol i c.

aL~tituto per ta Nutrizione delle Piante, 1012.5 Torino, Italy. bDip.di (;him. [no,~. FL*. Mater., via P.Giuda 7, 10125 Jbffno, Itatv.CDip. di Chim. Ino,R. MetalL, via .enezlan 21, 20133 Milano, Italy.

Pero.'fidases are hemoproteins that catalyze the oxidation of a large number of sub-

strates [1]. The previous studies on the purification and characterization of the major

cationic peroxidase have been reported [2]. On a more recent preparation of the zucchini

peroxidase we have been able to improve the purification and better characterize the

major anionic and cationic isoenzymes.

The principal proteic fractions (ZPOA

anionic and ZPOC cationic) were first

purified by ion-exchange cromatography

as described previously [21. then ~'

preparative isoelectric focusing. Both ZPOA and ZPOC optical and

CD spectra are analogous to those of

corrispondent horse radish isoenzymes,

(HRPA anionic and HRPC cationic ),

with the Soret band at ,~ = 403 nm (Fig. 1).

The two major fractions,

checked by SDS page and gel isoelectric

focusing, show one ~lomogeneous band

(M.W.38.800 : pl 3.8 ) and two bands

(M.W.41.200; pI 9.2 and M.W. -40.000; pI

9.0) respectively. Optical spectro-

photometry was used to study their reactivity

against organic substrates in comparison

with that of a more common horse radisch

isoenzymes. The specific activity of ZPOA

and HRPA in phosphate buffer 0.1 M

against H202 and different substrates

(Table 1), was measured at pH 7 and 25°C.

FIGURE 1. Electronic spectra of

ZPOA ( ...... ) and HRPA ( - )

Substrate

DMAB MBTH

o-Dianisidine

Hydroquinone

Eazyme

HRPA

ZPOA

HRPA

ZPOA

HRPA

ZPOA

Activity.

(units/rag)

1701.0

316.6

178) 0

569.7

36Z5

7_.691.0

TABLE l. Specific activity of ZPOA, HRPA

We can observe the higher value for ZPOA / hydroquinone specific activity with respect

to that of HRPA / hydroquinone for equal enzyme and H202 concentration. 1. M.Morrison and G.R.Schonbaumm, Annu. Rev. Biochem., 45, 861 (1969).

2. L.Casella, M.Gullotti, A.Pintar and A.Marchesini, Bier'him. Biophys. Acta, 872. 216 (1986).