Comp. Biochem. Physiol. VoI. 78B, No. 2, pp. 499-505, 1984 0305-0491/84 $3.00 + 0.00 Printed in Great Britain © 1984 Pergamon Press Ltd

CHARACTERIZATION OF CATALASE FROM HYALOMMA DROMEDARH CUTICLE*

RAGAA R. HAMED Biochemistry Laboratory, National Research Centre, Dokki, Cairo, Egypt

(Received 11 October 1983)

Abstract--l. Extracts of the tick Hyalomma dromedarii cuticle and its haemolymph contain both catalase (EC 1.11.1.6) and superoxide dismutase (EC 1.15.1.1).

2. The enzyme catalase has been purified from the cuticle of the tick to apparent homogeneity. The molecular weight of the enzyme was estimated to be 230,000 + 6000 and the subunit to be 60,500 + 2500.

3. The pH optimum for catalatic activity is between 6.5 and 8 whereas the pH optimum for DOPA oxidation is over 8.5.

4. The enzyme had a Km for H202 for catalatic activity of 8.6 mM and for DOPA oxidation of 8.3 mM. 5. The enzyme will oxidize a number of o-dihydricphenols (catechol, 4-methylcatechol, DOPA,

dopamine and gallic acid). 6. The Km for DOPA oxidation by cuticle catalase was 4 mM identical to that of liver catalase. 7. The possible relationship between cuticle catalase activity and hardening of the cuticle is discussed.

INTRODUCTION

The presence of catalase, superoxide dismutase, per- oxidase and glucose oxidase in locust cuticle was reported (Candy, 1981). The proposed role for glu- cose oxidase in cuticle is to generate hydrogen per- oxide that may function as an oxidizing agent. Re- cently catalase was purified from the developing embryo of t t . dromedarii ticks. The purified enzyme oxidized o-diphenots, with absolute requirements for H20 2 (Kamel and Hamed, 1982).

Many cuticles contain enzyme activities that will oxidize diphenols, phenol oxidases (Ishaaya, 1972; Yamazaki, 1969; 1972; Barrett and Anderson, 1981), and peroxidases (Locke, 1969 and Locke and Krish- nan, 1971). Also, involvement of catalase in the process of cuticle formation has been suggested (Schildknecht et al., 1970; Schildknecht, 1971 and Kamel and Hamed, 1982).

Despite the biological importance of catalase in hardening of the cuticle as well as protecting the cell against H202 it has never been isolated from the cuticle of the arthropods. The present investigation described a method for the purification and charac- terization of catalase from H. dromedarii cuticle. The relation between catalase and hardening of the cuticle is discussed.

MATERIALS AND METHODS

Fully engorged adult female H. dromedarii were collected from camels at market near Giza, the abdomens detached and the abdominal cuticle isolated by the method described

*This study was assisted by agreement No. 03-051-N between the National Research Centre, Dokki, Cairo, A.R.E., and Institute of Allergy and Infectious Diseases (National Institute of Health), Bethesda, MD, USA.

tAbbreviations used: DOPA, 3,4-dihydroxyphenylalanine; dopamine, 3,4-dihydroxyphenylethylamine; EDTA, ethylenediaminetetraacetic acid; SDS, sodium dodecyl sulphate; TEAE, triethylaminoethyl.

by Hackman and Goldberg (1971) for unhardened insect cuticle.

Tissue extract of H. dromedarii abdominal cuticle was prepared by homogenization using a Sorvall Omnimixer in 0.1 M phosphate buffer pH 7.0 for at least 1 hr: extract was centrifuged at 5000g for 20 min at 5°C.

Enzyme assays

Catalase (EC 1.11.1.6) activity was determined by mea- suring decomposition of hydrogen peroxide spec- trophotometrically at 240nm using a Varian spec- trophotometer series 634. Catalase units were calculated as described by Liick (1965). Activity was expressed in inter- national units (one IU is that amount of enzyme con- suming 1 #mole H202/min at 25°C) and specific activity as units/ms protein.

Unless otherwise stated o-dihydricphenol oxidase type activity was routinely assayed using DOPA as substrate.

DOPA,t catechol, 4-methylcatechol, dopamine and gal- lic acid oxidation products were followed at 475, 480, 490, 465 and 400 nm respectively in reaction mixture containing 50raM sodium pyrophosphate buffer (pH 8.0), 0.5mM MnCI 2, 10 mM H202, 5 mM o-dihydricphenol and suitably diluted enzyme concentrations. Assay reaction mixture in- cluding all but catalase was used as control. One unit o-dihydricphenol oxidation by catalase was that amount of enzyme sufficient to cause an O.D. change of 0.1/min.

Phenol oxidase activity was determined according to Anderson (1978). The coloured reaction product of the oxidized product of 4-methylcatechol and glycylglycine was followed at 470 nm in reaction mixture containing 10 mM 4-methyl catechol, 10 mM glycylglycine, 0.2 M sodium ace- tate buffer pH 5.5 and 0.1~o Naz-EDTA and suitably diluted enzyme concentration. The reaction mixture was incubated at 40°C for 30min and the absorption at 470nm was recorded.

Peroxidative activity: peroxidative activity of catalase in which guaiacol or o-dianisidine were used as substrates was followed spectrophotometrically at 470 and 460nm, re- spectively, according to the methods of Kamel et al. (1977) 1 ml vol: 50#moles phosphate buffer pH 7.8, 0.1 #mole EDTA, 0.01/~mole ferricytochrome c, 0.05/~mole xanthine assayed in terms of its ability to inhibit the superoxide- mediated reduction of ferricytochrome c by the xanthine oxidase system. The assay reaction mixture contained in a

499

500 RAGAA R. HAMEI)

1 ml vol: 50#moles phosphate buffer pH 7.8, 0.1 #mole EDTA, 0.01 ,umole ferricytochrome c, 0.05/~mole xanthine and suitably diluted xanthine oxidase to produce rate of reduction of ferricytochrome c at 550 nm of 0.025 absorb- ance units/rain. Inclusion of enzyme extract containing superoxide dismutase caused inhibition of ferrocytochrome formation. The amount of superoxide dismutase required to inhibit the rate of reduction of cytochrome c by 50~ (0.0125/rain) at 25C is defined as I unit of activity (McCord and Fridovich, 1969).

Protein determination

Protein was determined according to the method of Lowry et al. (1951), using bovine albumin as a standard.

Mol. wt determination

The mol. wt of H. dromedarii cuticle catalase was deter- mined using gel filtration (Andrews, 1964). Ultrogel ACA 34 column (1.6 cm i.d. × 97 cm) in 0.05 M sodium phosphate buffer pH 7.0 was calibrated with blue dextran, crystalline liver catalase, y-globulin, bovine albumin and ovalbumin. Protein solutions (2 ml) were applied to the same column and eluted at a flow rate of 8 ml/hr.

Polyacrylamide disc electrophoresis

The method of Davis (1964) was used for polyacrylamide gel electrophoresis. Subunit mol. wt was determined by SDS disc get electrophoresis as described by Weber and Osborn (1969). SDS-denatured ~,-globulin L, bovine serum albu- min, ovalbumin, 7-globulin H, cytochrome c and ribo- nuclease were used as reference standard. Protein bands were located by staining with Coomassie brilliant blue.

Purification q[' H. dromedarii cutich, catalase

Unless otherwise stated all purification steps were per- formed at 4 -7C. Crude extract was prepared by homoge- nization of 200 H. dromedarii thick cuticles in 0.05 M Na phosphate buffer pH 7.0. The homogenate was centrifuged at 5000g for 10min and the supernatant "crude extract" was saved for further purification.

The crude extract was applied to a column of TEAE cellulose previously equilibrated with 0.05 M phosphate buffer pH 7.0. Catalase activity did not exchange with the column material and appeared in the void volume. Fraction containing catalase activity were pooled.

Calcium phosphate gel, 4ml (0.3!'; solid content) was prepared according to Keilin and Hartee (1938) and slowly added to the pooled fraction from the TEAE cellulose column with continuous mixing, samples were withdrawn and tested for catalase activity after centrifugation in order to check for complete adsorption of the enzyme. The gel was collected by centrifugation at 5000g for 5 rain, and sus- pended in 3 ml of 30°0 ammonium sulphate solution. Most of the activity was recovered in the supernatant after centrifugation.

Calcium phosphate gel eluate was placed on top of gel filtration column (Ultrogel ACA 34, dimensions 1.6 cm × 97 cm) previously equilibrated with 0.05 M phos- phate buffer pH 7.0. The material was eluted with the same buffer, at a flow rate of 8 ml/hr.

RESULTS AND DISCUSSION

Catalase and supero.vide dismutase actirities in cutich, and haemolymph (![ H. dromedar i i

Catalase and superoxide dismutase activities were measured in cuticle extract and haemolymph of the tick H. dromedarii. Specific activities of catalase and superoxide dismutase were found to be 49 and 24.1 in the cuticle extract and 6.0 and 15 in the hae- mo lymph respectively. Catalase and superoxide dis- mutase are generally considered to have a role in protect ing cells against the toxic effect of the hydro- gen peroxide and the superoxide anion. However detailed studies of location or character iza t ion of these enzymes in cuticle were not reported. No phe- nolase activity could be detected using the method described by Anderson (1978) in cuticle extract. The presence of hydrogen peroxide and superoxide anion ( 0 2 ) dur ing au toxida t ion of o-dihydr icphenols (Cohen and Heikkila, 1974), and the occurrence of o-dihydr icphenol ic compounds and DOPA in the blood and cuticle of various insects is well established (Pryor et al., 1949: Pryor, 1955).

Enzyme purfh'cation

The procedure of the purification of H. dromedarii cuticle catalase is given in Table 1. The procedure involves ch roma tog raphy on T E A E cellulose, cal- cium phospha te gel and Ultrogel ACA 34. The initial specific activity in the crude cuticle extract was 49 uni ts /mg protein. The enzyme was not bound to T E A E cellulose and emerged in the void volume (Fig. 1). Pooled fract ions were assayed for catalat ic and D O P A oxidizing activities. Both activities were eluted together with a rat io of 1.3. The specific activity of the calcium phospha te gel eluate increased 44.6-fold over the crude extract. The purity index (405/280 nm absorbance ratio) of the calcium phospha te gel eluate was 0.35. Gel f i l tration on Ultrogel ACA 34 increased the specific activity 229.5-fold over the crude extract with a purity index of 0.6 (Fig. 2). However, the DOPA:ca ta l a se rat io did not change. The value of 0.4 has been reported for the purity index of purified H. dromedarii egg catalase (Kamel and Hamed, 1982). The purified enzyme was pure as judged by disc electrophoresis (Fig. 3).

Spectral analysis

The absorp t ion spectrum of H. dromedarii cuticle catalase exhibited two maxima at 280 and 405 nm which cor respond to a protein 280 nm and a Soret at 405 nm. The rat io of the absorbance at 405 nm to tha t at 280 nm was 0.6 for the purified enzyme (Fig. 4).

Table I. Purification scheme of H. dromedarii cuticle catalasc

Total protein Total units

(rag) a b

Crude extract 280.0 l 3,724 17,978.0 TEAE eluate 79.2 13,500 17,280.0 Ca phosphate gel eluate 5.6 12,400 16,606.0 Ultrogel ACA 34 eluate 0.54 6106 7937.88

Specific activity Purification Recovery % a b a b a b Ratio*

49.0 64.2 100 100 I.M 170.4 218.0 3.5 3.4 98 96.1 1.28

2187.0 2935.0 44.6 45.8 90 91.4 1.34 11250 14,699.6 119.5 228.9 44 44.1 1.30

*Specific activity of DOPA oxidation divided by catalatic specific activity, a, catalatic activity: b. DOPA oxidation

Catalase from Hyalomma dromedarii cuticle 501

0.6

0.4

0.2

100 200 300 400 500 600 700

_0.05 M iNa phosphate buffer

I, ,0.I M Na Cl - ~ - D - ~ _ 0 . 5 M Na ¢1

£

E t~

.13

..Q

. <

Eluate ( ml )

100

80 ~

60 "E

,>_ 40 ~

20

Fig. 1. Elution profile for the chromatography of crude H. dromedarii cuticle extract on TEAE cellulose column (1.6 cm i.d. x 60 cm) using different molarities of NaC1 in 0.05 M sodium phosphate buffer pH 7.0

for elution: absorbance at 280 nm, ----; catalase activity, O . . . . O.

Mol. wt determination

The mol. wt of the purified catalase was estimated to be 230,000 + 6000 ( +__ SE, N = 3) from the elution volume on Ultrogel ACA 34 and the subunit weight to be 60,500 _+ 2500 by SDS-polyacrylamide disc gel electrophoresis (Fig. 5).

Mammal ian and bacterial catalases exhibited mo- lecular weights ranging from 230,000 to 250,000 (Deisseroth and Dounce, 1970): however, the presence

of higher and lower molecular weights (Schildkenech et al., 1970; Kamel and Hamed, 1982; Baird et al., 1977; Claiborne et al., 1979) was also reported.

Effect o f p H on catalatic activity and DOPA oxidation

The effect of pH on catalase activity was examined between pH 5 and 10 using 0.05 M acetate buffer for pH 5-5.8, 0 .05M sodium phosphate buffer for pH 6.2-7.8 and 0.05 M sodium pyrophosphate buffer

E t "

E

.13

.O <

0 . 2 - -

0.1-

o I 0 50

,,f i I

1_~___,__L¢7 100

Eluate (mr)

A 150 200

800

6OO

zOO

200

¢--

3 v

> ,

Fig. 2. Elution profile for the chromatography of calcium phosphate gel eluate on Ultrogel ACA 34 column (1.6 cm i.d. x 97 cm) using 0.05 M sodium phosphate buffer for elution: absorbance at 280 nm,

. . . . , catalase activity, × . . . . x .

502 RAGAA R. HAMED

@ OR1GIN

1 2 ~)

Fig. 3. Diagram of disc electrophoresis of the purified H. dromedarff cuticle catalase. 1. Polyacrylamide gel electro- phoresis of 50 #g of purified catalase. 2. Polyacrylamide gel

electrophoresis in presence of SDS.

for pH 8.4-10 (Fig. 6). The optimal pH activity of H. drornedarii cuticle catalase was between pH 6.5 and 8 for H202 decomposition and pH 9 for DOPA ox- idation.

Michaelis constants (K,,) of H. dromedarii cuticle catalase

Decomposition of H202 by H. dromedarii follows typical Michaelis-Menten kinetics up to at least 10raM with a Km value of 8.6raM for catalatic activity and 8.3 mM for DOPA oxidation activity (Fig. 7). These values are in good agreement with the values reported for the H. dromedarii egg catalase. The calculated K~ values for DOPA for both cuticle and liver catalase were found to be 4 mM (Fig. 8).

Oxidation o f o-dihydricphenols by H. dromedarii cuticle catalase

Oxidation of catechol, 4-methylcatechol, DOPA, dopamine and gallic acid by H. dromedarii cuticle catalase were followed at 480, 490, 475, 465 and 400nm respectively. Relative activities considering DOPA as 100 were found to decrease in the order gallic acid (468), dopamine (221), 4-methylcatechol (72) and catechol (21). No peroxidative activity could be detected when o-dianisidine or guaiacol were used as substrates at pH 5.6, 7.0 and 8.0. Purified catalase from H. dromedarii developing embryos (Kamel and

c rd

<~

0.6

0.5

0.4

0,3

0.2

0.1

C700 '

1 l

s / / . . . . •

I II

/ / s J

I I I , , 1 600 500 400 300

W . L ( n n 7 )

Fig. 4. Absorption spectrum of purified H. dromedarii catalase ( - - ) and liver catalase ( . . . . ) in 0.05 M sodium phosphate buffer pH 7.0.

Catalase from Hyalomma dromedarii cuticle 503

2 ° , ~ -

2 . 2 -

2.0

1.8

1.6

1.4

1

1 2 2

3 3

I I 4.5 5 5.4

Log motecular weight

Fig. 5. Estimation of the mol. wt of H. dromedarii cuticle catalase and its subunit. Plot of log mol. wt vs elution volume/column void volume (ordinate) determined by gel filtration and relative mobility

(abscissa) by SDS-polyacrylamide gel electrophoresis.

Hamed, 1982) and P. notatum (Kamel and Hamed, 1983) exhibited the same properties.

Proteins isolated from the tick Boophilus microplus (Acarina ixodidae) abdominal cuticle differed quite markedly from the proteins isolated from Scylla serrata (Decapoda: Portunidae) and Agrianoma spi- nicollis (Coleoptera: Cerambycidae) in amino acid composition (high alanine content), molecular weight distribution, isoelectric point (higher) and solubility.

The peculiar properties of the Boophilus proteins may be a reflection of the ability to undergo a rapid stretching (Hackman, 1974). Also, the lowest values reported for chitin are 11.2 and 3.8700 in highly stretchable abdominal cuticle from Rhodnius and Boophilus (Hackman, 1975): however, chitin amounts usually to between 20 and 50% of the procuticle. It could be a general property of unsclerotized cuticles that they are plasticized at low pH values and it is

lOe

80

._> 6O

r~

N aO r r

20

/ / / /

/ / J ,/ I I . . . . "f" I t I 5 6 7 8 9 10

pH

Fig. 6. Effect of pH on H. dromedarii cuticle catalase activity, ----; DOPA oxidation, O . . . . O.

Km = 8.6 mM E

o !.5

o ;.0 I ©

-6

0.5

2> 0.2 0.1

1/H202 ( raM)

I I I I I 0.I 0.2 0.3 0.4 0 5

1/H202 (m M) Km=e.3mM

6 -

1 ~ - J i [ J I l i

-3.4 -0,2 0 0.2 0.4 0.6 0.8 1.0

Kin: 4rnM

Fig. 7. Lineweaver-Burk plot relating H. dromedarii cuticle catalase catalatic activity and DOPA oxidation activity to hydrogen peroxide concentration.

E

O

E

-4"

2 r~

(5

5d" I I . . . . l J I

-(3.25 0 0.25 0,5 0.75 1.0

504 RAGAA R. HAMED

I /OOPA mM

Fig. 8. Lineweaver-Burk plot relating H. dromedarii cuticle (a) and liver catalase (b) DOPA oxidation activity to DOPA concentration.

Catalase from Hyalomma dromedarii cuticle 505

possible to obtain the opposite effect if the pH is changed too much.

Oxidation o-dihydricphenolic compounds by cata- lase may play a role in hardening of the cuticle and, since plasticity is regulated in vivo through hor- monally controlled changes in intracellular pH (Rey- nolds, 1975), catalase may be controlled the same way.

Acknowledgement--The technical assistance of Miss Elham Desoky is greatly appreciated.

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