A novel 8.7 kDa protease inhibitor from chan seeds (Hyptis ... - UAM

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84 C. Aguirre et al. / Comparative Biochemistry and Physiology Part B 138 (2004) 81–89 Fig. 1. Zymogram of the crude extract in native PAGE. A: Crude extract of chan (H. suaveolens) seeds, B: Amaranth trypsin inhibitor used as positive control. (1993) using casein as substrate. For protease activity on X-ray film, we used the method described by Machhandra and Manavendra (1994). 2.3.2. Protease inhibitor activity determination Inhibitor activity against trypsin and trypsin-like enzymes of P. truncatus was assayed by monitoring the initial rate of hydrolysis of BAEE at 253 nm in 0.15 M Tris–HCl, CaCl2 0.05 M, pH 8.1 after 3 min of incubation (Schwertz and Takenaka, 1955). Inhibitor activity against proteases of T. castaneum, C. maculatus, A. obtectus and S. zeamais was assayed by monitoring the hydrolysis of casein at 280 nm in 0.04 M succinic acid, 0.06 M NaCl, pH 6.5 after 3 min of incubation (Kakade et al., 1969). At pH 2.5, it was assayed by monitoring the hydrolysis of hemoglobin at 280 nm in 0.2 M citric acid, 0.1 M NaCl, after 3 min of incubation (Lenney, 1975). One unit of proteolytic activity was defined as an increase in 0.01 absorbance units under the assay conditions described. Inhibitor activity was defined as the difference between the proteolytic activity measured in the absence and in the presence of the inhibitor. Inhibition Units (IU) were calculated as follows: IUymls Enzyme ODyŽ EnzymeqInhibitor. OD 0.01=Sample Ž ml. Samples of the inhibitor obtained from the purification procedure were screened for inhibitory Fig. 2. Gel filtration chromatography after (NH ) SO precip- 4 2 4 itation. Fractions were monitored for absorbance at 220 nm (line) and trypsin inhibitor activity (diamonds). activity against trypsin, chymotrypsin, papain and elastase. 3. Results 3.1. Purification When subjected to electrophoresis and stained for inhibitory activity, crude extract of Chan flour contained a single protein inhibitor (Fig. 1). Proteins were precipitated and separated by gel filtration (Fig. 2). The peak containing the inhibitory activity was collected and resolved by ion Fig. 3. Ion exchange chromatography of the fraction obtained after gel filtration. Fractions were monitored for absorbance at 220 nm (line), trypsin inhibitor activity (diamonds) and NaCl gradient (broken line).
C. Aguirre et al. / Comparative Biochemistry and Physiology Part B 138 (2004) 81–89 85 Table 1 H. suaveolens trypsin inhibitor (HSTI) purification Procedure Specific Yield Purification activity (%) fold (IUymg protein) Crude extract 138 100 1.00 (NH 4) 2SO4 156 84 1.14 precipitated fraction G-75 Filtration 441 50 3.21 fraction Ion-exchange 3800 61 27.80 fraction RP-HPLC 7900 10 57.40 fraction Fig. 4. RP-HPLC chromatography of the fraction containing the ion-exchange chromatography fraction containing inhibitor. The asterisk indicates the peak corresponding to inhibitor activity. Fig. 6. HSTI isoelectric point determination was 3.4. Fig. 5. Purification steps of H. suaveolens trypsin inhibitor (HSTI) isolated from chan (H. suaveolens) seeds; M: Molecular mass markers, A: Crude extract, B: Precipitated fraction, C: G-75 chromatography fraction, D: ion-exchange chromatography fraction, E: RP-HPLC. exchange chromatography (Fig. 3). The inhibitory activity eluted as a single peak. However, the fraction containing the inhibitory activity after ion exchange chromatography was heterogeneous, containing minor non-inhibitory proteins, which were removed by a final step of reverse phasehigh performance liquid chromatography (Fig. 4). From the two protein peaks obtained, the major peak contained the inhibitory activity, which was homogeneous by SDS-PAGE (Fig. 5). The recovery and relative fold purification at different stages of purification are given in Table 1.
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A novel 8.7 kDa protease inhibitor from chan seeds (Hyptis ... - UAM

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