INTRODUCTION Peroxidase is a ubiquitous class of enzymes, whose primary function is to oxidize a variety of hydrogen donors at the expense of hydrogen peroxide (2). The soybean plant includes 15-20 peroxidase isozymes. It was determined by activity stained isoelectric focusing gels (2). The soybean seed coat is a unique tissue in the soybean plant. It has only one peroxidase isozyme (2). The soybean seed coat soybean peroxidase (SBP) has a pI value of 4.1 and a molecular mass of approximately 37 kilodaltons (1).
SBP is a relatively new peroxidase, which is attractive as a biocatalyst for two reasons. One is thermostability. SBP is stable from 20°C to 90°C. Another one is pH stability. SBP is stable from pH 2 to pH 9(5). Commercial preparations such as Sigma have low activity, so we were interested in developing a method to quickly purify SBP so we could study SBP using the wide range of spectroscopic and electrochemical methods used to study peroxidases in our lab.
MATERIALS AND METHODSThe following reagents were obtained commercially as analytical grade or better and were used as received: potassium dibasic phosphate (Aldrich), potassium chloride (Fisher Scientific), PEG 600 MW (Aldrich), 30% hydrogen peroxide (Fisher Scientific), and pyrogallol (Sigma).
Soybean Peroxidase was purified from commercial Chinese soybeans "Montsew" which were obtained from the Sun Sun Chinese market (Boston, MA). Soybean seed coat was obtained from soybeans, which were soaked in the water for about four hours. Then the soybean seed coat was blended with extraction buffer 50 mM KH2PO4, pH 7.0 in a ratio of 4 mL of buffer per gram of seed coat. Then 600 MW PEG was added to give a final concentration of 12% (w/v). The coat was homogenized to form an aqueous two-phase system (7). Then gauze cloth was used to separate the liquid and solid. The liquid was concentrated by Amicon using a YM10 filter five times using distilled water from an initial volume of 50 mL to a final volume of 10 mL. Then samples were filtered using 0.45 µm syringe filters prior to HPLC. HPLC was performed a BioCAD/SPRINT using a PerSeptive DEAE Poros (4.6 mm/100 mm) column.
The specific activity of the purified SBP was measured optically in 100 mM potassium phosphate monobasic buffer pH 6 containing 30% (w/w) hydrogen peroxide and 0.5% (w/w) pyrogallol at 20°C using the standard Sigma peroxidase assay. Optical absorbance measurements were made on a HP8452A UV-visible diode array spectrophotometer using 1.0 cm path length rectangular supracil cuvettes (Hellma).
RESULTS AND DISCUSSIONThe peroxidase was purified by anion exchange high performance liquid chromatography using 50 mM KH-2PO4, pH 7.0, and a linear gradient (0-500 KCl) on a DEAE Poros (4.6 mm/100 mm) column.
Figure 1 shows a representative chromatogram for unpurified soybean seed coat SBP extract. The protein was obtained by collecting fractions corresponding to the peak marked "*" in Figure 1.
Fractions were pooled using ultrafiltration with a YM10 (Amicon). The salt was removed by washing five times with distilled water from an initial volume of 50 ml to a final volume of 10 ml. 100 µl of this final solution were re-injected into the HPLC. The result is shown in Figure 2.
Next, a loading study was performed to determine how much SBP could be purified in one run. Figure 3 shows the results for 20 µl, 50 µl and 100 µl injections. It shows that every time an increasing of SBP was injected, the peak shape was not affected. Therefore, up to an injection volume of 100 µl could be purified in one run.
Finally, the purified SBP was pooled and lyophilized on a Virtis vacufreeze unit overnight. Then the dry SBP powder was assayed for catalytic activity using the standard Sigma HRP assay in which pyrogallol (substrate) is oxidized by peroxidase to purpurogallin, a highly colored product. The formation of this product is monitored by UV-Vis spectroscopy at 420 nm at 20°C. The initial linear increase in absorbance with time is taken as a quantitative measure of the catalytic activity of the peroxidase. A larger specific activity is usually taken to imply a more active enzyme. The purified SBP had activity of 36.11±2.69 unit/mg (see Table 1). The activity is three times higher than that forthe unpurified SBP from Sigma. The specific activity of this SBP can be further improved by applying the method developed by Shakya and Chen (5).
ACKNOWLEDGEMENTSWe want to thank the NSF (MCB 598680), ACS Project SEED, and the Millipore Foundation. We also want to thank the Northeastern Section of the American Chemical Society for support of this work. During this experience, I want to thank Dr. PAM Mabrouk (Professor at Northeastern University), who always taught and support me. I also want to thank Dr. Qing Deng for help. Finally I would like to thank Ross Edwards who works at PE Biosystem. He always helps me to solve the problems.
REFERENCES1. Gillikin, J. W.; Graham, J. S. Plant Physiol. 1991, 96, 214-220. "Purification and Developmental Analysis of the Major Anionic Peroxidase from the Seed Coat of Glycine max."2. Huangpu, Jin.; Graham, Myrna c.; Graham, J. S. Plant Gene Register PGR 95-136. "Cloning of a soybean cDNA encoding the abundant anionic seed coat peroxidase."3. Vreeke, Mark S.; Yong, Khin Tsun and Heller. Analytical Chemistry. 1995, 67, 4247-4249. "A Thermostable Hydrogen Peroxide Sensor Based on Wiring of Soyben Peroxidase."4. Wang, Bingquan.; Li, Bin.; Wang, Zhenxin.; Xu, Guobao.; Wong, Qun and Dong, shaojun. Analytical Chemistry. 1999, 71, 1935-1939. "Sol-Gel Thin-Film immobilized Soybean Peroxidase Biosensor for the Amperometric Determination of Hydrogen peroxide in Acid Medium."5. Shakya, S.; Chen, Z.; Mabrouk, P.A. "An Improved Method for the Purification of Soybean Peroxidase." URL: http://clearinghouse mwsc.edu/
UNPURIFIED SPECIFIC ACTIVITY
SOYBEAN SEED HULLS
PURIFIED SPECIFIC ACTIVITY
MEAN VALUE ± STARDARD DEVIATION
26.95 ± 4.4
36.11 ± 2.69
FIGURE 1 CAPTIONFigure 1. Purification of SBP by anion-exchange chromatography at pH 7. Chromatographic conditions: 50 µm DEAE, 4.6 mm/100 mm anion exchange resin column (PerSeptive), detector wavelength, 280 nm; flow rate 5 ml/min, 0.05 M potassium phosphate buffer, pH 7, 0-500 mM KCl gradient (24.6 column volume).
FIGURE 3 CAPTIONFigure 3. Loading study. Chromatograms were obtained for 100 µl, 50 µl, and 20 µl injections of a 1 mg/ml SBP sample. Chromatographic conditions: 50 µm DEAE, 4.6 mm/100 mm anion exchange resin column (PerSeptive), detector wavelength, 280 nm; flow rate 5 ml/min, 0.05 M potassium phosphate buffer, pH 7, 0-500 mM KCl gradient (24.6 column volume).
FIGURE 2 CAPTIONFigure 3. Loading study. Chromatograms were obtained for 100 µl, 50 µl, and 20 µl injections of a 1 mg/ml SBP sample. Chromatographic conditions: 50 µm DEAE, 4.6 mm/100 mm anion exchange resin column (PerSeptive), detector wavelength, 280 nm; flow rate 5 ml/min, 0.05 M potassium phosphate buffer, pH 7, 0-500 mM KCl gradient (24.6 column volume).