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Characterization of a peroxidase in excretory-secretory product of adult Paragonimus westermani
Y B Chung,1Y Kong,1S Y Cho,*1S Y Kang,1B C Choi,2 and H S Lee2
1Department of Parasitology, College of Medicine, Chung-Ang University, Seoul 156-756, Korea.
2Department of Biochemistry, College of Medicine, Chung-Ang University, Seoul 156-756, Korea.
Received June 30, 1993; Accepted July 21, 1993.
Abstract
When activity of peroxidase in adult Paragonimus westermani was monitored using o-dianisidine and H2O2 as substrates, its specific activity was 1.5 times higher in excretory-secretory product (ESP) than in crude extract. The enzyme was purified by two purification steps of Sephacryl S-300 Superfine gel permeation and DEAE-Trisacryl M anion exchange chromatographies. Its activity increased 16.9 fold with 32.3% recovery. The enzyme was inhibited totally by 1 millimoles of dithiothreitol (DTT), 2-mercaptoethanol and azide. Molecular mass was 16 kDa in reducing SDS-polyacrylamide gel electrophoresis (PAGE) or 19 kDa in TSK-Blue gel filtration high performance liquid chromatography (HPLC), respectively. Special staining for peroxidase by diaminobenzidine on SDS-PAGE confirmed the activity. The peroxidase was less reactive to a paragonimiasis serum when observed by SDS-PAGE/immunoblot. In addition, specific activities of superoxide dismutase (SOD) and catalase were also identified in the ESP. High activities of these antioxidant enzymes in ESP indicate that they are parts of defense mechanisms against reactive oxygen intermediates from host.
Figures
Fig. 1 Elution profile of ESP of adult P. westermani in a Sephacryl S-300 Superfine gel permeation chromatography (1.6 ×70 cm). The column was equilibrated with 0.01 M PBS (pH 7.4 containing 0.1 mM EDTA and eluted with the same buffer at a flow rate of 20nl/hour·cm2. Allocation of 1.8 ml were assayed for enzyme activities. • : Protein content,. Inverted arrowheads indicate the respective enzymes. Bar (-) indicates the pooled fraction of respective enzyme. ▪ : SOD activity, ▵ : Catalase activity, □ : Peroxidase activity.
Fig. 2 Elution profile of peroxidase in ESP of adutl P. westermani on DEAE-Trisacryl M anion exchange chromatography. Partially clarified enzyme solution containing high activity of peroxidase was applied to a 1.6 × 5 cm sized DEAE-Trisaryl M columnand eluted through 1.02 M Tris-HCl buffer (pH 7.2) containing 0, 0.02, 0.05. 0.1 ir 0.2 M stepwise salt concentration. activity, inverted arrow indicate the salt gradient. Bar (-) indicates the pooled fraction containing high peroxidase activity.
Fig. 3 Determination of molecular mass of perocidase by TSK-Blue analytical gel filtration HPLC. Ten microgram of the enzyme was eluted through the column at a flow rate of 1 ml/minute. Standard proteins used were alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa) and cytochrome c (15.4 kDa). Inverted arrow (↓) indicates the retention time of the peroxidase.
Fig. 4 SDS-PAGE findings of the purified peroxidase on 7.5~15% gel in reducing condition. Throughout the electrophoresis, constant current of 30 mA was supplied. The gel was stained with Coomassie blue R-250 and destained with methanol/acetic acid solution. Mr: Molecular mass in kDa, Lane 1 showing the crude ESP. Lane 2 exhibits Sephacryl S-300 filtered fraction and lane 3 demonstrates the purified peroxidase.
Fig. 5 Finding of the purified enzyme in special staining for peroxidase. Separating gel of 10~15% was used for portein separation in reducing condition. After SDS-PAGE, the gel was soaked in 0.01 M potassium phosphate buffer (pH 7.2) containing 2 mM o-dianisidine 2 mM 3,3-diaminobenzidine and stained with 0.1 mM H2O2 and Cr: crude extract of adult P. westermani, P: Purified peroxidase from the ESP.
Fig. 6 SDS-PAGE/immunoblot using a confirmed patient serum to the ESP, adult worm extract and the purified peroxidase showing a typical finding. After transfer the resolved proteins to nitrocellulose paper by electrophoresis at 100 V for 2 hours at 4℃, a patient serum, diluted at 1:150 in PBS/T, was reacted overnight as a probe. M2: Molecular mass in kDa, Cr: Crude ESP, P: Purified peroxidase, A: Whole worm extract of adult P. westermani.
Tables
Table 1 Comparison of activities of peroxidase, SOD and catalase in ESP and crude extract of adult P. westermani
Table 2 Purification of peroxidase in ESP of adult P. westermani
Table 3 Effect of inhibitors on peroxidase activity in ESP of adult P. westermani
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