Peroxidase is an important class of heme enzymes that catalyze the oxidation of a wide range of organic substrates and the reduction of hydrogen peroxide. An important relatively new plant peroxidase is soybean peroxidase. (SBP) The 37 kDa enzyme, derived from soybean hulls, is acidic (pI 4.1) and contains one heme unit. SBP has two important characteristics that make it of much current interest in biocatalysis. The first is its` remarkable thermostability. Depending upon the pH, SBP can withstand temperatures between 20 ºC to 90.5 ºC without denaturing. The second useful characteristic of SBP is its’ pH stability. SBP is stable and catalytically active at pH values between 2 and 8. SBP exhibits maximum catalytic activity at pH 2.4. Most heme enzymes denature easily under such extreme temperature and pH conditions.
We are extremely interested in spectroscopically characterizing this new peroxidase. The SBP commercially available exhibits low catalytic activity and contains several contaminants. Therefore, we wish to report an improved method for the purification of SBP by anion-exchange high performance liquid chromatography, which produces SBP with a markedly higher catalytic activity.
MATERIALS AND METHODS
The following reagents were obtained commercially as analytical grade or better and were used as received: 2-[N-Morpholino]ethanesulfonic acid buffer (Sigma), NaCl (Fisher Scientific), pyrogallol (Sigma), H2O2 (Fisher Scientific) and potassium monobasic phosphate buffer (Aldrich).
Soybean peroxidase was obtained from Sigma and Organic Technologies and was used as received unless otherwise noted. SBP was purified using PerSeptive Sprint Biocad perfusion chromatography instrument with a 50 mm DEAE, 10 mm X 50 mm anion exchange resin column (PerSeptive). Ultrafiltration with a YM30 (Amicon) was used to remove the salt; the purified protein was washed 5 times with distilled H2O from an initial volume of 10 ml to a final volume of 1ml. Finally the SBP was lyophilized on a Virtis Vacufreeze unit overnight.
The specific activity of the purified SBP was measured colorimetrically for the oxidation of 42.3 mM pyrogallol to purpurogallin in 0.01 M potassium phosphate buffer, pH 6.0, in the presence of 7.84 mM hydrogen peroxide at 20 0C. 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 DISCUSSION
Purification of SBP was accomplished by anion exchange chromatography. Dual wavelength detection at 280 nm and 406 nm was used to distinguish SBP that was intact from SBP that which was denatured. Intact protein was expected to have a heme active site chromophore, which absorbs at 406 nm. All protein whether intact or denatured absorbs at 280 nm due to the aromatic amino acid residues.
First, we investigated the effect of pH on the separation. Figure 1 shows the chromatograms of SBP at pH 5 (top), 6 (middle), and 7 (bottom). The best separation of the 4 major eluting components can be seen at pH 6 (middle). The largest peak, representing intact SBP, elutes at 375 s and has absorbance at both wavelengths. At pH 6, the separation of the intact protein from the denatured protein eluting at 220 s and 490 s is maximized. Therefore the best separation occurs at pH 6.
Next the effect of salt (0 – 1.5 M NaCl) was investigated on the separation at pH 6. Chromatogram A in Figure 2 (pH 6 and 0.75M NaCl) displays the best resolution between the pure SBP peak at 345 s and the impure components at 220 s and 490 s.
SBP was next purified and fractions containing the purified protein were pooled. The purified protein was re-injected at the set conditions. The bottom chromatogram in Figure 2 shows the proof of purity as indicated by the presence of only one large peak at 345 s. The purified protein was characterized by a Rz value of 2.5.
Conservation of time is an important factor in HPLC. Thus a loading study was carried out. Figure 3 displays the chromatograms obtained for increasing sample load (10 ml, 50 ml, and 100 ml of 1 mg/ml SBP solution in distilled H2O). The best resolution is seen in chromatogram B (50 ml injection).
Finally the flow rate for the method was optimized. The original flow rate of 10 ml/min was decreased to 5 ml/min. This increased the run time from 15 min to 30 min. The chromatogram obtained had approximately the same resolution as the one at 10 ml/min. Therefore the 10 ml/min flow rate which results in a 15 min run time was considered optimal.
Since SBP is regarded as a potentially useful biocatalyst, the catalytic activity of the purified SBP was examined. Table 1 compares the specific activity of purified SBP to that obtained from two commercial sources Sigma and Organic Technologies. The specific activity for the purified SBP samples was determined using the standard peroxidase colorimetric assay in which pyrogallol is oxidized to purpurogallin, which absorbs at 420 nm. The purified SBP shows markedly higher catalytic activity than unpurified SBP obtained from the two commercial sources. Specifically, the purified SBP is 166 % more active than the unpurified Sigma SBP and 57 % more active than the SBP obtained from Organic Technologies.
We thank Organic Technologies for the gift of the SBP used in this work. S. S. is extremely grateful to Jeff Bernard (PE Biosystems), Sivashankar Sivakolundu and Pamela Hallock for help and support. This work was supported by NSF CAREER Award (MCB 598680) to P.A.M.
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Catalytic Activities of SBP from Different Sources
(purified, this work)
(n = 3)
FIGURE 1 CAPTIONFigure 1. Purification of SBP by anion-exchange chromatography at pH 5 (A), 6 (B) and 7 (C). Chromatographic conditions: 50 mm DEAE, 10 mm X 50 mm anion exchange resin column (PerSeptive), detector wavelength, 406 nm; flow rate 10 ml/min, 0.05 M 2-[N-Morpholino]ethanesulfonic acid buffer, 0 – 0.75 M NaCl gradient (15 column volume).
FIGURE 2 CAPTION
Figure 2. Proof of purity for SBP. Chromatogram (A) at pH 6, 0-0.7 5 M NaCl gradient (15 column volume). Chromatogram (B) obtained by reinjection of pooled fractions obtained from 320 s – 370 s for repeated runs. Chromatographic conditions: 50 mm DEAE, 10 mm X 50 mm anion exchange resin column (PerSeptive), detector wavelength, 406 nm; flow rate 10 ml/min, 0.05 M 2-[N-Morpholino]ethanesulfonic acid buffer.
FIGURE 3 CAPTION
Figure 3. Effect of increasing sample loading. Chromatograms were obtained for 100 ml (A), 50 ml (B) and 10 ml (C) injections of a 1 mg/ml SBP sample. Chromatographic conditions: 50 mm DEAE, 10 mm X 50 mm anion exchange resin column (PerSeptive), detector wavelength, 406 nm; flow rate 10 ml/min, 0.05 M 2-[N-Morpholino] ethanesulfonic acid buffer, 0 - 0.75 M Nacl gradient (15 column volume).