Biochimica et Biophysica Acta, 1120( 19921 167-172

167

© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/~.~2/$05.00

BBAPRO 34151

Purification and characterization of a soluble peroxidase of rat preputial gland: comparison with lactoperoxidase Prabir K. De and Ranajit K. Banerjee Depatlmen! of Physiology. Indian Institute of Chemical Biology. Calcutta (htdia)

(Received Z'i June 1991) (Revised manuseriptreceived4 November 1991)

Key words: Preputialgland peroxidase:Lacloperoxidase;Enzymepurification;Peroxidase:(Rat) A highly active ,soluble peroxidase (donor: H202 oxidoreductasc EC I.i !.i.7) has bccn purified from the preputial gland of the rat by hydroxylapatitc chromatography, ammonium sulfate fraclionation, Sephadex gel filtration and affinity chromatography on con A-Sepharosc. The enzyme shows apparent homogcneity when analysed by acid and alkaline-PAGE. Its molecular, spectral, kinetic and catalytic properties were compared with those of bovine lactoperoxidase (LPO). Preputial gland pcroxidase (PPO) is a glycoprotein of molecular weight of 711-73 kDa slightly lower (78 kDa) than that of LPO. Using isoelectric focussing, PPO was resolved into eight different closely spaced protein species spanning a pl range of 5.4 to 6.4, while LPO focusscs into several closely spaced protein bands in the pl range 8.5-9.3. PPO is similar to LPO in its spectral (Soret) and some kinetic properties, but it differs significantly from LPO in substrate (H,_O2) tolerance and substrate inactivation. PPO also differs from LPO in showing differential inactivation by SDS. Both enzymes arc glycoproteins and although concanavalin A (con A) showed a variable interaction with both enzymes, wheat germ agglutinin interacted specifically with LPO only. We suggest that PPO, the ~cretory peroxidase of the preputial gland, differs significantly from LPO in its molecular and catalytic properties.

Introduction The preputial gland of the rat of both sexes consists of a pair of unusually enlarged modified sebaceous ectodermal exocrine glands opening to the exterior on either side of the urethral meatus. The glands have the characteristics of an accessory sex gland and are highly sensitive to various sex hormones [1,2]. A number of exocrfne glands of ectodermal origin are characterized by the presence of secretory soluble peroxidase. These includ4,• salivary glands [3], mammary glands [4], lacrimal glands and bovine Harderian glands [5]. Recently we identified a highly active soluble peroxidase in the rat preputial gland [6]. The enzyme is secreted in the sebum and some catalytic and immunological properties of the crude enzyme were similar to those of uterine fluid peroxidase [6]. We have observed by

Abbreviations: ConA,concanavalinA; WGA,wheat germ agglutinin; PPO, preputial gland peroxidase;LPO, lactoperoxidase;SDS. sodium dodecylsulfate; PMSF, phenylmethylsulfonylfluoride;PAS, periodic acid Schiff. Correspondence: R.K. Banerjee, Department of Physiology,Indian Institute of Chemical Biology, 4 Ra!:: S.C. Mullick Road, Calcuna 700032, India.

Western blotting that all soluble rat peroxida~s including the preputial gland enzyme crossreact with lactoperoxidase antiserum and have a molecular weight in the range 73-80 kDa [7]. In earlier histochemical studies, the peroxidases of rat salivary, lacrimal and lactating mammary glands were termed as 'iactoperoxidase' [8-11], implying close identity to bovine lactoperoxidase. Bovine lactoperoxidase is immunologically identical to the peroxidases from bovine salivary, lacrimal and Harderian glands [3-5]. The pcroxidases of sheep and goat milk as well as human saliva are also immunologically similar to bovine lactoperoxidase [12-19]. Morrison et al. showed that purified pcroxidase from bovine submaxillary glands [3] is also identical with milk peroxidase [4]. Recently human salivary peroxidase was shown to be both similar to and dissimilar from lactoperoxidase in some of its molecular, catalytic and kinetic properties [15,16]. Although some controversy exists as to the nature and origin of the peroxidase in human milk and colostrum [17,18], Langbakk and Flatmark [19] recently claimed to have identified a classical iactoperoxidase in such material. The term 'lactoperoxidase' may be a misnomer when applied beyond the milk system. The generalization that all soluble peroxidases of mammalian ectodermal glands and their secretions are lac-

20

168 toperoxidases remains unproven, in this paper we present evidence that rat preputial gland peroxidase has some spectral and kinetic similarity with bovine iactoperoxidase, but that major differences exist in molecular characteristics including isoelectric ( p l ) values and isozyme patterns, as well as in some catalytic properties in HzO 2 requirement, in SDS-inactivation and in differential interaction with lectins. These results indicate that the soluble peroxidase of the rat preputial gland is not identical with bovine lactoperoxidase.

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Materials and Methods

Chanicals Lactopcroxidase from bovine milk (RZ, A4u/A,.s,, = 0.88), phenylmethylsulfonyl fluoride (PMSF), amethyl-mannoside, con-A-Sepharosc, concanavalin A (con A), whcatgerm agglutinin (WGA), sodium dodecyl sulfate (SDS), molecular weight markers for SDSPAGE and Pharmalyte 3-10 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Bio-gel HTP (hydroxylapatitc) was obtained from Bio-Rad Laboratories (Richmond, USA). Scphadex G-150, molecular weight markers for gel-filtration and p l markers for isoelectric focussing were procured from Pharmacia (Uppsala, Sweden). All other reagents used were of analytical grade purity.

Purification of peroxidase from rat preputial glands Twenty-two pairs of glands from female rats (2(X) g) were homog,:nized at 0-4°C in a Potter-Elvehjem glass homogenizer in 2[).0 ml of 10 mM potassium phosphate buffer, pH 8, containing 0.25 M sucrose and 1 mM PMSF. The soluble supernatant was passed through a column (1.5 × 16 cm) of hydrox~ylapatite equilibrated with 10 mM phosphate buffer, pH 8 at 4°C with a flow rate of 5 ml per h. Eiution was carried out with the same buffer containing 1 mM PMSF. The eluate which contained the bulk of peroxidase activity, was subjected to ammonium sulfate fractionation. The precipitate obtained between 45-70% saturation was dissolved in 1.0 ml of 20 mM Tris-HCI, pH 7.5, containing 151) mM NaC! and ! mM PMSF, and subjected to gel filtration at a flow rate of 8 ml per h on a Sephadex G-150 column (2.81 cm) equilibrated with the same buffer. Fractions containing peroxidase activity (eluted at VJV,, = 1.55) were pooled and the NaCI concentration was made 500 mM. The pooled fractions were then loaded onto a column of con-A Sepharose (! • 6 cm), previously equilibrated with 20 mM Tris-HCI, pH 7.5, 500 mM NaCI and 1 mM each of CaCI 2, MgCI 2 and MnCI 2. When the 230 nm absorption in the washings had reached a value close to zero, ! ml of 0.5 M a-methylmannoside-containing buffer was passed

15 >1 I¢

20

25

0 8ml froctlons - -

>1

Froctlon number

Fig. i. Affinity purification of the PPO on con A-Scpharose. Fraction I represents the start of the enzyme loading. Washing buffer conrained 20 mM Tris-HCI. pll 7.5 containing 500 mM NaCI. Specific elution was done by ~-methylmannoside (I).5 M) in washing buffer.

through the column at room temperature. Peroxidase was eluted with (I.5 M a-methylmannoside-containing buffer at a flow rate of 9 ml per h. The enzyme emerged in a sharp peak (fractions 18-23), as shown in Fig. 1, and was dialyzed against appropriate buffers or concentrated by lyophilisation as required.

Assay of peroxidase acth'ity Peroxidase activity during purification was assayed using KI as electron donor [6,7]. The catalytic activity of the isolated enzyme was later studied using guaiacoi as electron donor [201. When LPO was compared with PPO, the assay system contained 13 mM guaiacol plus 27 ~ M H 2 0 -, and enzyme in 50 mM Tris-HCI buffer, pH 8. All assays were carried out at 30 + 2°C.

Determination of k t The velocity constant (k~) for reaction of PPO with H 202 was determined at 300C according to the method of Chance and Maehly [21]. The reaction mixture contained (in a final volume of 3 ml): 13 mM guaiacol, 2.7 ~tM H 2 0 z and 4 - 1 0 -~ M PPO in 50 mM Tris-HCI buffer, oH 8.

Polyacrylamide gel electrophoresis Acid [22], alkaline [23] and SDS-PAGE [24] were carried out as described in the references. Protein was stained with Coomassie brilliant blue G-250 (0.25%) for 3 h at 37°C before destaining. To stain glycoprotein, the alkaline P A G E gels were washed overnight in 50% methanol-7% acetic acid solution and incubated in I% periodic acid and 7% acetic acid for 60 rain at 4°C in the dark followed by a similar incubation with Schiff's reagent. The gels were washed in 1% sodium metabisulfite containing 0.1 N HCI. Ovalbumin and bovine serum albumin were used as positive and negative controls, respectively.

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Molecular weight estbnation The molecular weight of the native PPO was estimated by gel filtration [7] and by sucrose density gradient centrifugation [25]. The molecular weight of the PPO was estimated using the equation of Martin and Ames [25] with LPO (78 kDa) as standard. The molecular weight of PPO, after denaturation by SDS in p r e ~ n e e of /~-mereaptoethanol, was determined [24] from a plot of relative mobility (Rf) against molecular weight of standard marker proteins.

P, •

lsoelectric focussing After dialysis against deionised water for 24 h, isoelectric focussing of purified preputial peroxidase, lactoperoxidase and a mixture of standard p l markers was carried out in a 5% acrylamide gel containing Pharmalyte (pl 3-10) as described in LKB instruction sheet 1818P. Peroxidase isozymes were stained for activity using diaminobenzidine and H202 [26]. Results

Yield and actirity of preputial peroxidase As summarized in Table !, purified preputial peroxidase showed a specific activity of I 1 253 ! ~ units with a 124-fold purification compared with the initial supernatant. Using molar extinction coefficients of 26 340 for l.~ [27] and 5570 for tetraguaiacol [28], PPO catalyzes the formation of 1281 p, mol 13- min[ mg -t and 456 /zmol tetraguaiacol min - ~ mg- ~ protein. Molecular characteristics in both alkaline and acid PAGE, the enzyme shows a single band and the activity stain also corresponded with the protein stain (Fig. 2). A positive PAS stain showed the peroxidase to be a glycoprotein. Using isoelectric focussing over the pH range 3-10, purified enzyme showed at least eight different closely spaced protein bands spanning the p l range from 5.4 to 6.4 (Fig. 3). All eight protein species (lane 1) showed peroxidase activity (lane 2). LPO (lane 3), focussed under identical conditions, showed a number of closely spaced bands spanning a p i range of 8.5-9.3, confirming its cationic character [29]. in gel chromatography,

i

°

J Fig. 2. Polyacrylamide gel electn~phoresis of the native PPO. (a) alkaline PAGE, glycopn~tein (PAS) stain (15 ~ug of pcroxidase); (h) alkaline PAGE, peroxidase activity stain (5/.tg); (c) alkaline PAGE, protein stain (211 tzg): (d) acid PAGE, protein stain ( 15 #g).

purified peroxidasc showed a molecular weight of 73 kDa (Fig. 4a). When denatured enzyme was subjected to SDS-PAGE, it showed a major band with a molecular weight of 73 kDa (Fig. 4b). A minor band of molecular weight 46 kDa was sometimes disccrnable. Pure LPO showed a single 78 kDa band with slightly lower mobility than PPO. On sucrose density gradient centrifugation (not shown) using LPO as rcfcrcnce protein, the mobility of PPO was slightly lower than that of LPO. Substituting the molecular weight of LPO (78 kDa) into the equation of Martin and Ames [25], a value of 70.2 kDa for PPO was obtained, which was close to that of 73 kDa determined by gel filtration and SDS-PAGE.

Spectral properties The purified PPO had a RZ value (A412/A28 o) of 0.41. Native enzyme showed a Soret band at 412 nm and minor peaks at 506, 536 and 576 nm. The Soret

TABLE !

Purification of peroxidasefrom preputial gland

105000X g supcrnatanl Hydroxylapatite unadsorbed fraction 45-70% (NHa)zSO4 precipitate Sephadex G- 150 eluate Con A Sepharose eluate

Total activity (units)

Total protein (mg)

Specific activity (unit.mg ; )

(Yield ~ )

Fold of purification

871111 8458 76511 5 304 4850

96 14.3 5.5 2.167 0.431

911.62 591.46 1 39 i 2447.6 I i 253

I1111 97.2 87.9 60.9 55.7

I 6.52 15.34 27 124.17

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pH

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