Homology of Plant Peroxidases

Abstract

Antisera specific for the basic peroxidase from horseradish (Amoracea rusicana) were used to examine homology among horseradish peroxidase isoenzymes and among basic peroxidases from root plants. The antisera cross-reacted with all tested isoperoxidases when measured by both agar diffusion and quantitative precipitin reactions. Precipitin analyses provided quantitative measurements of homology among these plant peroxidases. The basic radish (Raphanus sayius L. cv. Cherry Belle) peroxidase had a high degree of homology (73 to 81%) with the basic peroxidase from horseradish. Turnip (Bransica rapa L. cv. Purple White Top Globe) and carrot (Daucus carota L. cv. Danvers) basic peroxidases showed less cross-reaction (49 to 54% and 41 to 46%, respectively). However, the cross-reactions of antisera with basic peroxidases from different plants were greater than were those observed with acidic horseradish isoenzymes (30 to 35%). These expements suggest that basic peroxidase isoenzymes are strongly conserved during evolution and may indicate that the basic peroxidases catalyze reactions involved in specialized cellular functions. Anticatalytic assays were poor indicators of homology. Even though ho-mology among isoperoxidases was detected by other immunological methods , antibodies inhibited only the catalytic activity of the basic peroxidase from radish. Plant peroxidases (EC 1.1 1.1.7) are important in diverse cellular functions such as lignin biosynthesis, hormone generation, and detoxification of hydrogen peroxide. These enzymes are glycopro-teins composed of a single polypeptide chain, and they contain ferriprotoporphyrin IX as a prosthetic group. Each plant has numerous peroxidase isoenzymes that differ in substrate specificity and localization within the plant. In addition, isoperoxidases within some species and those from different species exhibit size heterogeneity. Isoenzyme mol wt range from 30,000 to 50,000 daltons. Comparative studies of root plant peroxidases have been performed with horseradish (12) and turnip (15) isoenzymes by peptide mapping. These studies provide only a qualitative estimate of homology between peroxidase isoenzymes. Primary structure comparisons have not been made, because only a basic isoenzyme from horseradish has been completely sequenced. Other studies of homology among peroxidases have focused on catalytic and phys-icochemical properties of the isoenzymes (1 1). These probes have not yielded quantitative estimates of homologies among root plant isoperoxidases. The purpose of the present study was to determine the relationships among horseradish peroxidases and basic isoperoxidases from a number of root plants by immunological methods. Anti-body specific for a basic isoenzyme of horseradish was used as a probe ofhomology among these plant peroxidases. Immunological methods were chosen, because these techniques provide quantitative measurements of homology and their application is simple. MATERIALS AND METHODS Horseradish Peroxidases. Horseradish peroxidase isoenzymes were purchased from Sigma Chemical Co. Two acidic isoenzymes (types VII and VIII) and a single basic isoenzyme (type IX) were homogeneous when assessed by SDS-PAGE1 (14) and were used without further purification. Isoelectric points for each isoenzyme were determined by focusing in a polyacrylamide gel containing ampholytes that gave a pH 3 to pH 10 gradient (10). Enzyme concentrations of horseradish peroxidase solutions were determined spectrophotometrically. The A of a 1 mg/ml solution at 403 nm is 2.5 (13). Kinetic constants for horseradish peroxidases were determined by the method of Lineweaver and Burk (3). Peroxidases from Other Root Plants. Basic peroxidase isoen-zymes were purified from 1 kg each of turnip (Brassica rapa L., cv. Purple White Top Globe), radish (Raphanus sativus L., cv. Cherry Belle), and carrot (Daucus carota L., cv. Danvers). The roots were washed, sliced, and homogenized with 1 L of distilled H20 in a Waring Blendor. Homogenates were then filtered through four thicknesses of cheesecloth and Whatman No. 3 filter paper. The filtered aqueous extracts were brought to 50%o saturation with solid ammonium sulfate and centrifuged at 4,000g for 20 min. After the pellets were discarded, the supernatant fluids were brought to 85% saturation with ammonium sulfate and centrifuged for 20 min at 4,000g. The 4,000g pellets were suspended in 5 mm sodium phosphate (pH 8.0), dialyzed extensively against the same buffer, and placed on DEAE-cellulose columns (2.5 x 40 cm) equilibrated in the 5 mM sodium phosphate buffer. Flowthrough fractions were pooled, concentrated by ultrafiltration (Amicon PM-10 membrane), and placed on Sephadex G-100 columns (2 x 90 cm) equilibrated in 50 mm sodium phosphate (pH 7.5) containing 0.15 M NaCl. Fractions with peroxidase activity were pooled, concentrated by ultrafiltration, and used without further purification. The mol wt and homogeneity of the final products were assessed by SDS-PAGE. Measurement of Enzyme Activity. The reaction used to monitor the peroxidase activity of all isoenzymes was reduction of H202 (Merck) by the electron donor o-dianisidine (Sigma). Reaction velocities were determined spectrophotometrically by measuring the appearance of oxidized dianisidine at 460 nm (16). Activity measurements were done at room temperature in 0.1 M citrate buffer (pH 5.3). Antiserum Production. New Zealand White rabbits were immunized biweekly with 1 mg of the basic horseradish isoenzyme. For immunization, an aqueous solution of the basic isoenzyme (1 1 Abbreviation: SDS-PAGE, SDS-polyacrylamide gel electrophoresis. 28 HOMOLOGY OF PLANT PEROXIDASES mg in 1 ml) was emulsified with an equal volume of Complete Freund's adjuvant (Difco Laboratories). Inoculations were made subcutaneously at several sites on the animal. Bleedings were taken from the marginal ear vein 10 days after each inoculation. Antisera are identified by animal number followed by the bleeding number. Agar Difusion Analyses. Double diffusion in agar analyses were performed as described by Ouchterlony (8). Peroxidases and antibody diffused toward one another at room temperature in a 1% agarose medium containing 0.15 M NaCl. Precipitin Tests. Antiserum and varied amounts of plant per-oxidases were incubated for 48 h at 47°C. The immune precipitates that formed were collected by centrifugation, washed twice with 0.9%1 (w/v) NaCl, dissolved by adding 0.1 ml 0.1 N NaOH, and brought to a final volume of 0.5 ml with water. The. antibody content of the immune precipitates was determined spectropho