Human Salivary Peroxidase and Bovine Lactoperoxidase are Cross-reactive B. MANSSON-RAHEMTULLA, F. RAHEMTULLA', and M.G. HUMPHREYS-BEHER2 Department of Community and Public Health Dentistry, 1Department of Oral Biology, University of Alabama School of Dentistry, UAB Station, Birmingham, Alabama 35294; and 2Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, Florida 32610

Peroxidases are abundant in nature, and the primary function of mammalian peroxidases is to catalyze the peroxidation of halides and pseudohalides. Previous studies have shown that antibodies raised against bovine lactoperoxidase moderately cross-react with human salivary peroxidase, a feature that has been used in the present study to examine epitopes common to the antigen and human salivary peroxidase. Polyclonal antibodies against a highly purified preparation of bovine lactoperoxidase were raised in rabbits, and their properties were examined. In double-immunodiffusion experiments, the two enzymes showed partial identity, and in competitive radioimmunoassay and enzyme-linked immunosorbent assay, lactoperoxidase replaced the labeled and coated antigen, while salivary peroxidase did not. However, salivary peroxidase from human and rat saliva samples and the purified enzyme in its non-reduced, reduced, and de-glycosylated forms were recognized by these antibodies, as analyzed by Western blot analysis and immunodetection. The major activity of these antibodies was directed against the protein core of the antigen. Immunodetection of the peptide fragments of bovine lactoperoxidase and human salivary peroxidase revealed structural differences in the two enzymes. These antibodies also precipitated an in vitro translation product from rat-parotid-gland cell lysate that, on SDS-PAGE, compared favorably with the expected molecular weight of a de-glycosylated peroxidase. The antibodies partly inhibited the enzyme activity of salivary peroxidase and the peroxidase in rat parotid gland lysate, but the enzyme activity of lactoperoxidase was not affected by addition of antilactoperoxidase IgG between 25 and 400 [Lg/mL. The enzyme activity remained unchanged in all samples when pre-immune IgG was used. J Dent Res 69(12):1839-1846, December, 1990

Introduction. Several investigators have raised polyclonal antibodies in rabbits against bovine lactoperoxidase (LPO), but these antibodies have not been characterized in terms of their cross-reactivity to various peroxidases, their antigenic determinants, and species specificity. Morrison and Allen (1963) were the first to raise antibodies against LPO (anti-LPO) and to show that these antibodies cross-reacted with salivary peroxidase (SPO) from bovine salivary glands. Revis (1977) detected peroxidase in human parotid saliva through the use of heterologous crossreacting antibodies to LPO using immuno-electrophoretic techniques. Moldoveanu et al. (1982) demonstrated that anti-LPO cross-reacted with a component in human saliva and that this component stained positively with a peroxidase stain. However, the antibodies did not cross-react with the peroxidase Received for publication May 3, 1990 Accepted for publication August 2, 1990 This investigation was supported in part by USPHS Research Grants DE 07076 (to B. M.-R.) and DE 08778 (to M. H.-B.), by the Small Instrumentation Grant Program DE 08812, and by Biomedical Research Support Grant #7RR 05300-25.

present in human milk, and thus they concluded that the peroxidase in human milk was not identical with LPO or SPO. The catalytic inhibition of bovine LPO and rat submandibular peroxidase by the antiserum against LPO has been studied by Chakraborty and Hati (1986). They reported that LPO and rat peroxidase bound to the antiserum and demonstrated that the enzyme activity of LPO was inhibited by 55-60%, while no inhibition was detected for rat peroxidase. Studies from our laboratory have shown that anti-LPO cross-reacts with the peroxidase in human whole and parotid saliva, and that the crossreacting component is a polypeptide with a molecular weight of 78,000, as demonstrated by immunodetection on nitrocellulose (Mansson-Rahemtulla et al., 1984). These antibodies have served as a useful tool in large-scale preparation of human SPO and have obviated tedious and time-consuming conventional procedures of protein purification (Matnsson-Rahemtulla et al., 1988). Peroxidase enzymes are commonly found in mammalian tissues and secretions. Although they vary in molecular weight and function, all peroxidases, by definition, catalyze the oxidation of a substrate by decomposition of hydrogen peroxide (H202) to produce the oxidized donor and water (Thomas, 1985). In the presence of the appropriate co-factors, these enzymes have a wide range of biological activities, e.g., the peroxidation of halides and the thiocyanate ion (SCN-) yields products toxic to micro-organisms (for review, see Pruitt and Reiter, 1985). Other functions are de-toxification of reactive intermediates of oxygen reduction and de-toxification of H202 produced by many micro-organisms (Carlsson, 1987). Hydrogen peroxide, even in low concentrations (> 10 wxmol/L), has been shown to be toxic to mammalian cells (Hanstrbm et al., 1983; Tenovuo and Larjava, 1984). Salivary peroxidase and LPO were for several years considered to be identical with respect to their immunological and chemical properties; however, recent studies from our laboratory have shown that the two enzymes differ in their chemical composition and catalytic properties (Matnsson-Rahemtulla et al., 1988; Pruitt et al., 1988). For useful immunological tools to be created for immunocytochemistry and molecular biology as well as for structural differences between the related peroxidases to be studied, rabbit antisera were raised against a highly purified preparation of LPO, and the specificity of the elicited antibodies was examined.

Materials and methods. Saliva collection.-Collection of rat whole saliva was performed after stimulation with 5 mg pilocarpine (Sigma Chemical Co., St. Louis, MO) per animal (male Wistar rats, 175225 g), which had been anesthetized with 0.75 pLg/g body weight of sodium pentobarbital (Sigma Chemical Co.). Stimulated human parotid saliva was collected from healthy donors by use of Lashley cups (Lashley, 1916), as previously described (Pruitt et al., 1983). Tissue preparation. -Rat parotid glands were identified by gross morphology and were removed from animals that had been anesthetized with sodium pentobarbital (Sigma Chemical 1839

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Co.) and killed by exsanguination. The glands were homogenized at 40C in 50 mmol/L Tris/HCl buffer, pH 7.5, with a glass-homogenizer. The homogenate was sonicated for ten to 15 s and centrifuged in a Sorval RC 5B Centrifuge for one h at 40C and at 10,000 g (ra, 10.7 cm). The pellet was discarded and the peroxidase activity determined in the supernatant. The protein content of the supernatant was determined by the method of Bradford (1976) with bovine serum albumin (Fraction V, Sigma Chemical Co.) used as a reference. Preparation ofperoxidases. -Bovine LPO (purity index = A412/A280 = 0.76) was purchased from Sigma Chemical Co. and further purified by the procedure described by Paul et al. (1980). Briefly, LPO was dissolved in 2.0 mol/L sodium acetate, pH 7.0, and applied to a column (2.5 x 15 cm) of Phenyl-Sepharose (Pharmacia LKB Biotechnology, Piscataway, NJ) that had been equilibrated in the same buffer. After extensive washing with 2.0 mol/L acetate buffer, the bound material was eluted with a negative gradient of 2.0 mol/L 0.05 mol/L sodium acetate, pH 7.0, over a total volume of 500 mL. The LPO eluted toward the end of the gradient and had a purity index of 0.92. Human SPO was purified according to the procedure described by Mansson-Rahemtulla et al. (1988). The purification procedure involved sequential passage of lyophilized parotid saliva through an immuno-affinity column, cation-exchange chromatography, and repeated affinity chromatography on BlueSepharose CL-6B (Pharmacia LKB Biotechnology). The final product (2.25 mg of SPO obtained from 1 L of saliva) was homogeneous and had a purity index of 0.81. Enzyme assays.-Peroxidase activity was measured with 2,2'azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) used as a substrate (Sigma Chemical Co.). The assay conditions have been described by Shindler et al. (1976). For a typical assay, the sample (usually 25 [IL) was added to 2.0 mL of 1.55 mmol/L ABTS in 0.1 mol/L sodium-acetate buffer, pH 4.4, and the reaction was started by addition of 1.0 mL of 0.31 mmol/L H202. One enzyme unit is equivalent to an increase in absorbance of 32.4/min at A412 nm and corresponds to the amount of enzyme catalyzing the oxidation of 1 mmol/ L of the ABTS-substrate under the described conditions. This definition assumes an absorption coefficient of 32,400 mol-1 cm-1 for ABTS at 412 nm. The data were obtained from a Cary Model 219 spectrophotometer (Varian, Sugar Land, TX) interfaced with a PDP-11/73 computer system (Salcris Systems Promed, Birmingham, AL). Enzyme activities were calculated from slopes determined by least-squares analysis of measurements recorded (sampling rate, 360 s-1) during the first 10 s of the reaction. Preparation of antiserum.-Preparation of polyclonal antibodies to LPO has been described previously (Mainsson-Rahemtulla et al., 1984, 1988). Briefly, a highly purified preparation of LPO (450 Ag of protein dissolved in 0.5 mL of de-ionized water) was mixed with 0.5 mL of Freund's complete adjuvant (GIBCO, Grand Island, NY) and injected subcutaneously into several sites on the backs of female New Zealand white rabbits. Subsequent injections (450 jig of protein) in Freund's incomplete adjuvant (GIBCO) were made at 14-day intervals. Blood samples were collected prior to the first injection and seven days after each injection. The blood was allowed to clot for 30 min at room temperature, and for four h at 40C, after which the serum was separated by centrifugation. The appearance of antibodies directed against LPO was monitored by enzyme-linked immunosorbent assay. The IgG fraction from the rabbit antiserum was isolated by ammonium sulfate precipitation, as described by Garvey et al. (1983), and dialyzed extensively against borate-buffered sa-

J Dent Res December 1990

line. The concentration of IgG fraction was quantitated spectrophotometrically by the use of Al% = 13.8. Immunodiffusion. -Ouchterlony (1949) double-diffusion analysis was performed in 1% agarose matrix buffered with 10 mmol/L potassium phosphate and 150 mmol/L NaCi, pH 7.0. The antibody and antigen preparations were allowed to react in a humidified chamber for 24 h at 220C. The plates were dried and stained with Coomassie blue. Radio-immunoassay procedures (RIA). -Lactoperoxidase was iodinated with Na-125I-iodine (specific activity, 15.5 mCi '251/Ig iodine; Amersham, Arlington Heights, IL) by the chloramine T method (Sonoda and Schlamowitz, 1970), and protein-bound and free iodide were separated by chromatography on Sephadex G-25 (Pharmacia LKB Biotechnology). The specific activity of the iodinated protein was 2-3 jCi/gg. The standard binding assay used in the present study was as described by Caterson et al. (1979). A typical assay for the estimation of antibody titer to LPO contained an appropriate serum dilution in 0.1 mL of incubation buffer (0.5% deoxycholate, 0.1% NP40, 0.1% BSA, 0.15 mol/L NaCI, 0.03% NaN3, 0.01 mol/L sodium phosphate, pH 7.4) to which radio-iodinated LPO (approximately 40,000 cpm) in 0.1 mL of incubation buffer was added, and the mixture was incubated for 90 min at 220C. A 10% w/v suspension (0.15 mL) of heat- and formalin-inactivated S. aureus (IgGsorb, The Enzyme Center, Inc., Malden, MA) was added to each tube, and the mixture was shaken for 30 min at 22°C. The incubation was stopped by addition of 1.0 mL of wash buffer (0.25% deoxycholate, 0.1% NP-40, 0.15 mol/L NaCl, 0.1 mol/L Tris-HCl, pH 7.4) to the tubes. The S. aureus-bound antigen-antibody complex was pelleted by centrifugation at 1000 g for ten min at 22°C. The supernatant was discarded, the pellet washed twice with wash buffer, and the radioactivity determined by y-counting in a LKB-Wallac CliniGamma 1272 (Pharmacia LKB Biotechnology). Competitive binding assays were carried out as follows: The amount of antiserum (0.1 mL in incubation buffer) that would bind 50% of the radiolabel in the standard RIA was incubated with a mixture of radiolabeled LPO (20,000 cpm in 50 jiL incubation buffer), and increasing concentrations of unlabeled antigen or test substance in 50 [IL of incubation buffer were added, and the mixture was incubated at 22°C for 90 min. The percentage inhibition of binding was calculated relative to the amount of radiolabeled LPO that was bound in the absence of added unlabeled antigen or test substance (Caterson et al., 1979). Enzyme-linked immunosorbent assay (ELISA). -The ELISA assay used for the detection of specific antibody was a modification of the procedure described by Voller et al. (1976). Microtiter plates (Flow Laboratories, McLean, VA) were coated with 200 [IL/well of highly purified LPO (0.025 [g/mL) or SPO (0.250 [Ig/mL) in 20 mmol/L carbonate buffer, pH 9.6, and incubated for 16 h at 4°C. After the plates were washed with PBS-Tween (20 mmol/L sodium phosphate, 0.15 mol/L NaCl, and 0.05% Tween 20), they were blocked with 1% BSA in PBS-Tween for 90 min at 37°C. The plates were extensively washed with PBS-Tween, and appropriate antibody dilutions in the same buffer were added to the wells and incubated at 37°C for 90 min. The amount of antibody bound to the antigen was determined by addition of 200 [IL of a 1:3000 dilution of goat anti-rabbit IgG conjugated to alkaline phosphatase (Southern Biotechnology, Birmingham, AL) and was quantitated by hydrolysis of p-nitrophenylphosphate (Sigma 104R phosphatase substrate tablets, Sigma Chemical Co.). The color development was measured at 405 nm with a Titertek Multiscan ELISA plate reader (Flow Laboratories). To determine the ability of the purified antigen and test

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substance to interact with the specific antibody, a competitive ELISA assay was used. The microtiter plates were coated with a 0.025-pLg/mL solution of LPO or SPO (0.250 pLg/mL) in the carbonate buffer. To an appropriate antibody dilution, increasing concentrations of antigen or test substance were added and incubated overnight at 40C. After washing and blocking of the plates as described above, aliquots of the antigen-antibody/test substance-antibody complex were added to the wells, and the plates were incubated for 90 min at 370C. The remainder of the procedure was performed as described above. Antibody inhibition of enzyme activity.-The antibody inhibition of enzyme activity was determined for LPO, SPO, and cell lysate from rat parotid glands. To 100 [LL of the enzyme preparations (5 pg/mL in PBS, pH 7.0, containing 0.1% gelatin) and 100 4L of the cell lysate in 50 mmol/L TrisHCl (2.95 mg/mL protein), 100 p1L of increasing concentrations (50-800 pg/mL of IgG in PBS, pH 7.0) was added to each preparation, and the mixture was incubated for 30 min at 370C. After the incubation period, the samples were briefly vortexed, and each sample was divided into two fractions. To one fraction, 5 pL of a 10% suspension of heat- and formalininactivated S. aureus was added, and the mixture was incubated end-over-end for 60 min at 220C, while the other fraction was analyzed for enzyme activity by the ABTS method. After the incubation period, the samples were centrifuged for 15 min at 8800 g (ray, 4.5 cm) at 40C, and the supernatant was analyzed for enzyme activity. Two sets of control experiments were performed. In the first set, no antibody was added to the enzyme preparations and the cell lysate, while in the second set, 50-800 Rg/mL of pre-immune IgG was added, and samples were treated as described above. Electrophoretic techniques and immunodetection.-Slab gel electrophoresis, with 0.75-mm and 1.5-mm gels, was performed essentially as described by Laemmli (1970) and modified by Butler et al. (1981). Electrophoresis was conducted under both non-reduced and reduced conditions in either 515% gradient gels or 12.5% gels, and was for four h at 20 mA for 1.5-mm (14 cm x 17.5 cm) gel slabs and 45 min at 200 mV for 0.75-mm gels (7.5 cm x 10.0 cm). Immune-precipitated in vitro translation products were subjected to electrophoresis in 0.75-mm-thick (14.0 cm x 17.5 cm) 10% polyacrylamide gels by means of the modified Tris/glycine Laemmli system described by Pugsley and Schnaitman (1979), and subsequent x-ray fluorography was performed as described earlier by Humphreys-Beher et al. (1984). Samples were transferred to nitrocellulose (Schleicher & Schuell, Keene, NH) according to the procedure described by Towbin et al. (1979), as modified by Burnette (1981). After electrotransfer, immunodetection was performed by the procedure described by Larsson (1981). The nitrocellulose was incubated for one h at 220C in borate-saline buffer, pH 8.5, containing 1% BSA. Rabbit anti-LPO, 10 ug/mL in TBS, pH 7.5, containing 1% Triton X-100 was added and incubated for one h at 220C. After three washes in TBS-Triton, a 1:2000 dilution of horseradish-peroxidase-conjugated goat anti-rabbit antibody in TBS-Triton was added for 90 min followed by three TBS-Triton washes. The transfers were then incubated in 50 mL of a 0.5 mg/mL solution of diaminobenzidine (Sigma Chemical Co.) followed by addition of 15 AL of 30% H202 until brown reaction products were observed. The reaction was terminated by flushing with 5 mmol/L acetic acid. In control experiments, the nitrocellulose was incubated with pre-immune IgG and immunodetection was performed as described above. No reaction was observed when pre-immune IgG was used, showing that the substrate and H202 specifically reacted with the antibody-bound horseradish peroxidase and not the electrotransferred peroxidases.

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Protein modification and de-glycosylation.-Human SPO and bovine LPO were modified by boiling the samples for two min in the presence of 0.5% 3-mercaptoethanol. Deglycosylation of the two enzymes was accomplished by use of N-glycanase (Genzyme, Boston, MA), as described by Plummer et al. (1984). The peroxidases (10 pL, 2 mg/mL) were boiled for three min in the presence of 0.5% SDS and 0.1 mol/L P-mercaptoethanol. The samples were diluted with sodium phosphate buffer, EDTA, and NP-40 (final concentrations 0.2 mol/L, 0.005 mol/ L, and 1.25 mol/L, respectively). N-glycanase was added to a final concentration of 10 units/mL, and the reaction mixture was incubated for 16 h at 370C. Human transferrin served as a control to determine that the N-glycanase was active. Peptide maps by incomplete proteolytic digestion.-Partially digested LPO and SPO were compared by the method of Cleveland et al. (1977) with use of highly purified preparations of the proteins. The peroxidases (1 mg/mL) in 0.125 mol/L Tris-HCI, 0.5% SDS, 10% glycerol, pH 6.8, were boiled for 2 min. Aliquots of 10 pL of protein solution were incubated with 1 pL of each of the following: trypsin (grade V; 2 mg/ mL) (Sigma Chemical Co.), V8 protease from Staphylococcus aureus (1 mg/mL) (Miles Scientific, Naperville, IL), and subtilisin (0.25 mg/mL) (Sigma Chemical Co.) for 30 min at 37°C. Following addition of 2.5 pL1 of 10% SDS and 1.25 4L of 2mercaptoethanol, proteolysis was stopped when the samples were boiled for two min. Duplicate samples were analyzed on a 12.5% SDS-PAGE, and after electrophoresis the gel was cut into two parts. One part was stained with Coomassie blue, while the other part was used for Western blot and immunodetected as described above. Isolation of poly(A+)RNA.-Total gland RNA was isolated by the method of Chirgwin et al. (1979). Immediately on removal, 1 g of fresh tissue was homogenized in 4 mol/L guanidinium isothiocyanate, pH 7.0, containing 1.0% sodium sarkosyl, 50 mmol/L EDTA, and 25 mmol/L sodium citrate. The RNA was subsequently recovered by an 18-hour centrifugation through 5.7 mol/L cesium chloride at 35,000 rpm in a SW 50.1 rotor. Poly(A+) RNA was obtained from total RNA by the procedure of Aviv and Leder (1972) on oligo(dT)-cellulose. All the above reagents were ultra-pure and purchased from International Biotechnologies Incorporated (New Haven, CT). In vitro translation ofRNA. -Poly(A+) RNA was translated in vitro by the method of Pelham and Jackson (1976) with a reticulocyte lysate (nuclease-treated) prepared by Promega Corporation, Madison, WI. The reaction volumes used were 50 pL containing 0.5 pLg of normal or isoproterenol-treated parotid or submandibular-gland RNA. Incorporation of [35S]methionine (specific activity, 800 mCi/mmol; New England Nuclear; Boston, MA) into protein was determined by 10% trichloroacetic acid precipitation of 5 pL from translations in vitro on glass-fiber filters. Samples from translations were prepared for electrophoresis in SDS-PAGE and subsequent x-ray fluorography as described previously by Humphreys-Beher et al. (1984). Immune precipitation of in vitro translation products. -Immune precipitation of in vitro translations was performed by addition of 100,000 cpm of [35S]-methionine-labeled translation product to 1.0 mL of PBS containing 0.1% SDS, 0.5% sodium deoxycholate, and 0.5% Triton X-100 with 10 4L of antiserum to LPO or pre-immune serum. The mixture was incubated for 60 min on ice, after which Protein A Sepharose (Pharmacia LKB Biotechnology) was added, and the incubation continued for an additional 30 min. The sample was pelleted by centrifugation, washed three times in the PBS detergent buffer before final re-suspension in 25 pL of SDS gel sample buffer, and electrophoresed as described above.

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Results. Preparation of LPO specific antiserum. -Antibodies to LPO were detected in Ouchterlony double-diffusion experiments. The reaction of anti-LPO with LPO showed an immunological line of identity (Fig. 1). The same held true for the reaction of anti-LPO with SPO; however, SPO and LPO showed an immunological line of partial identity. These results indicate that LPO and SPO share one epitope, but that one of the antigens has an extra epitope that is recognized by the antibody. Tests were run with other dilutions of antibody and antigen, and the same results were obtained. The reactivity of the antigen to bind to the anti-LPO was tested with a competitive RIA. The binding of 1251-LPO was tested against different dilutions of antiserum, and the results indicated that a 1:6000 dilution was suitable for further binding studies. Displacement of 1251-LPO with unlabeled test substance was performed with this dilution of antibody. While it was found that 93.8 ng of LPO was required to achieve a 50% inhibition of the labeled antigen, 4000 ng of partially purified SPO did not displace any antibody-bound labeled LPO (results not shown). In further studies, the ability of purified SPO to displace the labeled antigen was tested. The results showed that high concentrations of the human enzyme did not compete with the labeled antigen. The antibody recognition was also tested in an ELISA. The results showed that a significantly higher concentration of SPO (0.250 pLg/mL) than LPO (0.025 Fxg/mL) was required for the microtiter plates to be coated so that reproducible standard dilution curves for both enzymes could be obtained. It was observed that LPO had a much higher affinity than SPO for binding to the antibody; while it was found that 3.6 vg/mL of IgG was required to achieve 50% binding for LPO, 21.7 ~ag/ mL was required for SPO (results not shown). The inhibition ELISA procedure was used to check crossreaction between LPO and SPO. The results are plotted as percent inhibition vs. concentration of inhibitor (Fig. 2). When the plates were coated with LPO (0.025 [tg/mL), 625 ng/mL of LPO was required to achieve a 50% inhibition. Under these conditions, it was observed that SPO exhibited only partial inhibition and that 10,000 ng/mL of SPO resulted in only 30% inhibition (Fig. 2A). For verification that anti-LPO IgG recognizes SPO, a second inhibition assay was performed in which

the plates were coated with 0.25 ,ug/mL of SPO, and this molecule was tested for its ability to compete with the binding of anti-LPO to the coated antigen. As can be seen in Fig. 2B, approximately 400 ng/mL of SPO and 1000 ng/mL of LPO were required for 50% inhibition. The inhibition assay was tested for its use in quantitation of peroxidase in saliva and for recognition of other peroxidases by the ELISA. Neither parotid saliva nor partially purified preparations of SPO showed inhibition of the binding of antibody to the coated LPO. The antibodies were also tested for their cross-reactivity to preparations of myeloperoxidase and horseradish peroxidase, and neither of these test substances was recognized by the antibodies as analyzed by ELISA. The inhibitory effects of the antibodies on the enzyme activity of LPO, SPO, and peroxidase in rat parotid gland lysate were tested by addition of increasing concentrations of antiLPO IgG. As shown in Fig. 3, panel A, the enzyme activities of LPO and rat parotid cell lysate were not affected by the antibodies. Only partial inhibition was observed for SPO, and approximately 300 Axg/mL of anti-LPO IgG was required for 100

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Double immunodiffusion test with rabbit IgG to bovine lactoFig. 1 peroxidase. Immunodiffusion was carried out for 24 h at room temperature as described in "Materials and methods". The wells contained 1 Pg of purified bovine lactoperoxidase (LPO) and 10 pLg of human salivary peroxidase (SPO). The center well contained 100 pig of anti-bovine lactoperoxidase IgG (Ab). -

Fig. 2 - Competitive inhibition enzyme-linked immunosorbent assay analyses depicting the ability of the antigens (bovine lactoperoxidase and human salivary peroxidase) to compete with the coated antigen for the rabbit anti-LPO IgG. (Panel A) The plates were coated with 0.025 ,g/rmL LPO. (Panel B) The plates were coated with 0.250 gug/mL of human SPO. In both experiments, the antibody concentration was 21.7 pLg/mL, and bovine LPO (W) and human SPO (U) at the concentrations indicated in the Fig. were used for competition. The optical density at 405 nm was measured 60 min after addition of the substrate.

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the maximum inhibition achieved, which was 40%. To evaluate whether a sufficient amount of IgG had been added to the enzyme preparations to bind all available antigen, a 10% suspension of Staphylococcus aureus that would bind a total of 700 FLg IgG was added. The results showed that 100 [Lg/mL of anti-LPO IgG was sufficient to bind all the available LPO (Fig. 3, panel B). In contrast, 200 [Lg/mL of IgG bound only 60% of the available SPO and 27% of the enzyme activity in the cell lysate. Furthermore, addition of IgG to SPO and rat parotid cell lysate did not have any effect on the binding properties or enzyme activity. The enzyme activity remained unchanged in all experiments when pre-immune IgG was used. Antibody-binding specificity to LPO, human parotid and rat whole saliva was studied by immunoblots of nitrocellulose to which protein bands from SDS-PAGE were electrophoretically transferred. As shown in Fig. 4, there was a clear immunodetection of LPO and peroxidases in both rat and human saliva.

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It should be noted that human saliva and rat saliva contain peroxidase at a concentration of 1-5 pLg/mL, and that in this experiment only an equivalent of approximately 0.2 mL of saliva was applied to the gel. In rat saliva, two bands migrating close together, both of slightly higher molecular weight than LPO, were detected. Three bands were detected in the parotid saliva sample, two of which migrated on the same level as LPO, while the third band had a higher apparent molecular weight than LPO. The higher-molecular-weight band was not stained with Coomassie blue in the electrophoresed parotid saliva sample; however, when a purified preparation of SPO was subjected to SDS-PAGE, all the three bands were stained with Coomassie blue and stained for enzyme activity by use of 3,3',5,5'-tetramethylbenzidine (Mansson-Rahemtulla et al., 1988). Additional analyses were made of the purified preparations of LPO and SPO. After electrotransfer of native, reduced, and deglycosylated LPO and SPO to nitrocellulose, the proteins were subjected to immunodetection. As can be seen in Fig. 5, antiLPO IgG cross-reacted with LPO and SPO in native, reduced, and de-glycosylated forms. These results indicate that the antibody recognition is not dependent on the conformation of the molecule, and that the N-linked oligosaccharides are not exclusively recognized by this antibody, but are (for the most part) directed against the peptide portion of the molecule. It should be noted that several high-molecular-weight bands were immunodetected in both non-reduced and reduced preparations of LPO; however, these high-molecular-weight bands were not stained with Coomassie blue. The high-molecular-weight bands are most probably aggregated forms of the enzyme that we and others observed previously (Matnsson-Rahemtulla et al., 1988; Makinen and Tenovuo, 1976). Similarly, in the non-reduced SPO prepa-

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43.0 30.0 21.0 1 4.4 Fig. 4 - Determination of antibody specificity to peroxidase enzymes by electrophoretic transfer to nitrocellulose and immunolocation analyses. Molecular weight markers (Lane A), bovine lactoperoxidase, 1 pug (Lane B), human parotid saliva, 100 jig (Lane C), and rat saliva, 100 ,ug (Lane D) were subjected to SDS-polyaciylamide electrophoresis on a 5-15% gel, electrotransferred to nitrocellulose, and the peroxidases localized by immunodetection as described in "Materials and methods". The IgG concentration was 10 pig/mL. Panel I depicts the Coomassie-Blue-stained gel and Panel II the immunoblots for the corresponding lanes.

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V8 PROTEASE

SUBTILISIN

Fig. 6 - Immunodetection of partially digested LPO and SPO. Onedimensional peptide maps of partial proteolytic digests of LPO and SPO were generated by incubating 10 IiL of LPO and SPO (1 mg/mL) with 1.25 pL1 of trypsin (1 mg/mL); 1.25 RL V8 protease (1 mg/mL) and 1.25 pL subtilisin (0.25 mg/mL) for 30 min at 37'C and subjecting the digests to electrophoresis on 10% SDS-polyacrylamide gels. The peptides were transferred to nitrocellulose and immunodetected with anti-LPO IgG used at a concentration of 10 ,ug/mL.

67.0 43.0 30.0

21.0 14.4

SPO LPO Fig. 5 - Reaction of anti-LPO IgG to reduced and de-glycosylated forms of LPO and SPO. The proteins (10 pLg) were untreated, treated with 0.5% 2-mercaptoethanol or digested with 10 units/mL N-glycanase, electrophoresed on a 5-15% SDS-polyacrylamide gel, electrotransferred to nitrocellulose, and immunodetected. The anti-LPO IgG concentration was 10 Pg/mL.

ration, a high-molecular-weight band was visualized by both Coomassie blue and immunodetection. We have previously shown that this band retains its enzyme activity (Mainsson-Rahemtulla et al., 1988). However, when SPO was electrophoresed under reduced conditions, this band could no longer be detected. Immunoreactive peptide fragments of LPO and SPO. -Onedimensional peptide maps of partial proteolytic digests of LPO and SPO were performed as described by Cleveland et al. (1977). The peptide patterns of LPO and SPO obtained after digestion with the proteases were significantly different, indicating differences between the two enzymes. A detailed description of the characteristic fragment maps of LPO and SPO will be published elsewhere (manuscript in preparation). Generally, proteolytic digestion of LPO resulted in a greater number of peptide fragments than the corresponding treatment of SPO. Immunodetection of fragments obtained after partial proteolysis is shown in Fig. 6. All trypsin-generated fragments were recognized by the antibody; however, the patterns were significantly different for LPO and SPO (Fig. 6, lanes 2 and 4). There were immunoreactive fragments present in LPO unique to this enzyme, e.g., two fragments that migrated with an apparent molecular weight of 30,000 and several low-molecular-weight peptides (lane 2). The major portion of the V8-generated LPO fragments were of high molecular weight and were recognized by the antibody (lane 6). The immunoreactive peptides of SPO differed from those of LPO; a distinct pattern consisting of eight SPO fragments was recognized by the antibody (lane 8). Digestion with subtilisin resulted in small-molecular-weight fragments for the LPO that were barely detected by the antibody, while under identical conditions, SPO was completely digested and no fragments were immunodetected (lanes 10 and 12).

In vitro translation. -So that the specificity of the antibody could be demonstrated, it was necessary to show that immunoprecipitation of a solution containing a mixture of radioactive proteins yielded a single product. Therefore, total poly(A+)-containing RNA was translated in reticulocyte lysate cell-free translation system, as described in the "Materials and methods" section. The rats were treated with isoproterenol for two, four, six, eight, and ten days, and this resulted in the expression of typical salivary proline-rich proteins. Fig. 7 shows the results of a typical experiment; lanes B to F demonstrate the presence of many labeled proteins in the total translation product. Immunoprecipitation of this mixture resulted in a single radioactive band (lanes H and K), while no radioactive material was obtained when preimmune serum was used (lanes G and J). The specificity of these antibodies was verified by the inclusion of 10 ,ug of unlabeled LPO in the immunoprecipitation reaction. Lanes I and L show the absence of radioactive products. The immunoprecipitated material had an estimated molecular weight of 72,000; in the experiment shown in Fig. 3, the apparent molecular weight of rat salivary peroxidase was 80,000. LPO and SPO preparations have been reported to have a carbohydrate content of 7-12% (Carlstrom, 1969; Mansson-Rahemtulla et al., 1988). If it is assumed that rat salivary peroxidase has similar carbohydrate content, then the translation product that was precipitated by the antibody compared favorably with the expected value of de-glycosylated peroxidase, the molecular weight of which would be in the range of 70,000-74,000.

Discussion. Previous studies have established that anti-LPO cross-reacts with the peroxidase present in human saliva (Revis, 1977; Moldoveanu et al., 1982; Mansson-Rahemtulla et al., 1984), an observation that has been used in the present study to elucidate the immunological properties of LPO and highly purified SPO. The detection of antigenic determinants unique to LPO and SPO has been useful for examination of epitopes common to the two peroxidases. The present report is the first to characterize the antibodies raised against a highly purified preparation of LPO, and in which specific differences in LPO and SPO antigenic determinants can be assigned.

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Vol. 69 No. 12

ANTIBODIES TO PEROXIDASES

cox 10

~31-

Fig. 7

-

Fluorogram of the cell-free translation products of mRNA from

rat parotid glands. Lanes B-F show the results of two, four, six, eight, and ten days of isoproterenol treatment. Lanes G and L show immunoprecipita-

tion; lanes G and J with pre-immune serum; and lanes H and K using antiserum to LPO; and lanes I and L are the results of the addition of 10 [Lg of unlabeled LPO to the immunoprecipitation reaction. The arrow indicates the position of the immunoprecipitated translation product.

For immunoreaction to occur between related proteins, there must be a 60% or greater sequence homology between the two

proteins (Cocks and Wilson, 1972). The cross-reaction of antibodies to bovine LPO with the human SPO substantiates that similarities in structure exist. However, the exact degree of homology between the two enzymes cannot be identified at this point. While the primary and secondary structures of LPO have not been determined, optical spectra and CD analyses have estimated 65% structure, 23% a-helix, and 12% unordered structure for LPO in aqueous solution (Sievers, 1979). The enzymeactive site of LPO has not been identified with certainty; however, Sievers (1980 and 1984; Sievers et al., 1983) has shown that the heme of LPO is a protoheme IX and that the proximal ligand of the heme iron may be histidyl imidazole. Nothing is known about the conformational structure and enzyme-active site of SPO. In studies reported elsewhere (Mansson-Rahemtulla et al., 1988; Pruitt et al., 1988), it was demonstrated that LPO and SPO differ in amino-acid and carbohydrate composition and in catalytic properties. In the present study, by means of the double-immunodiffusion technique, the anti-LPO showed a precipitin line of partial identity with SPO, confirming the observations by Moldoveanu et al. (1982) that antibodies raised against LPO reacted with the peroxidase in parotid saliva. The immunological differences between SPO and LPO were further demonstrated by the competitive RIA and ELISA. While LPO successfully competed with the antigen in both assays, SPO gave only partial inhibition when LPO was coated on the plate. However, when SPO was used for both coating of the microtiter plates and as a competitor, a typical inhibition curve was achieved. The anti-LPO reacted equally well with the reduced and native enzyme, and thus, we believe that the antibody recognizes a specific amino-acid sequence in the polypeptide chain of LPO and SPO, rather than a conformational epitope on these molecules. Elucidation of the immunological determinants) of a native glycoprotein is complicated by the presence of the potentially antigenic carbohydrate moiety. Any cross-reactivity between SPO and LPO could be due to carbohydrate. However, removal of N-linked oligosaccharides by N-glycanase did not affect the crossreactivity for either SPO or LPO, indicating that the antigenic determinants are present on the protein core of the enzymes. This observation was confirmed by the immunoprecipitation of the in

1845

vitro translated product that had an apparent molecular weight of a de-glycosylated peroxidase. After digestion of the enzymes with various proteases and use of the specific anti-LPO IgG to detect only the immunoreactive peptide fragments, almost no correspondence was seen between the fragments derived from LPO and SPO. However, both the digests from LPO and SPO contained immunoreactive fragments. Since distinct differences in the number and molecular size of these fragments were seen in both the trypsin and V8 digests, the purification and sequencing of these peptide fragments bearing the antigenic differences will be of interest in future studies so the differences in primary protein structure of LPO and SPO can be determined. Studies by Paul et al. (1986) have shown that a monoclonal antibody against bovine LPO does not affect the catalytic activity of the enzyme, while Chakraborty and Hati (1986) found that a polyclonal antibody to LPO inhibited the enzyme activity by 5560%. They also reported that this antibody bound to crude rat submandibular salivary peroxidase but did not inhibit its enzyme activity. In the present study, we have demonstrated that the polyclonal antibodies failed to inhibit the enzyme activity of LPO, while the enzyme activity of SPO and peroxidase found in rat parotid gland lysate was partially inhibited. These results indicate that the anti-LPO IgG used in this study recognizes different epitopes on LPO, SPO, and possibly the peroxidase found in the rat parotid gland. We had intended to develop a competitive RIA or ELISA for quantitation of peroxidase in human saliva using anti-LPO. However, because of the significant immunological differences between SPO and LPO shown in this study and in our previous report, such an assay would not have the specificity and sensitivity for clinical use. Furthermore, it has been shown by Ericsson and Ivarsson (1987) that endogenous antibodies to bovine LPO are common in serum of both children and adolescents. Although not tested in this study, it is highly probable that endogenous anti-LPO is also present in human saliva and that, therefore, these antibodies would interfere in an assay with polyclonal antibodies raised against bovine LPO. In summary, our results show that the antibodies raised against bovine LPO were moderately cross-reactive with SPO, a feature that has been useful for examination of epitopes on the protein core common to SPO and LPO. We have also demonstrated that these antibodies can be used to identify rat salivary peroxidase and in biosynthetic studies of rat salivary peroxidase.

Acknowledgments. We acknowledge the expert technical assistance of David C. Baldone and Bente Flatland and Diane Lovoy for the preparation of the manuscript. We also thank Drs. Jiri Mestecky and Kenneth M. Pruitt for fruitful discussions. REFERENCES AVIV, H. and LEDER, P. (1972): Purification of Biologically Active Globin Messenger RNA by Chromatography on Oligothymidylic AcidCellulose, Proc Natl Acad Sci USA 69:1408-1412. BRADFORD, M.M. (1976): A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding, Anal Biochem 72:248-254. BURNETTE, W.N. (1981): "Western Blotting": Electrophoretic Transfer of Proteins from Sodium Dodecyl Sulfate-Polyacrylamide Gels to Unmodified Nitrocellulose and Radiographic Detection with Antibody and Radioiodinated Protein A, Anal Biochem 112:195-203. BUTLER, W.T.; BHOWN, M.; DIMUZIO, M.T.; and LINDE, A. (1981): Noncollagenous Proteins of Dentin. Isolation and Partial

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