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Sweet potato defensin (SPD1) overproduced in E. coli (M15) was purified by Ni2+-chelate affinity chromatography. The molecular mass of SPD1 is about 8,600 Da determined by SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide gel electrophoresis). Our previous paper showed that SPD1 had antimicrobial, dehydroascorbate reductase and monodehydroascorbate reductase activities. The activity of SPD1 to inhibit angiotensin converting enzyme (ACE) was shown using N-[3-(2-furyl) acryloyl]-Phe-Gly-Gly (FAPGG) as substrate in a dose-dependent manner (27.56 ∼ 52.58 % inhibition). The 50% inhibition (IC50) of ACE activity required 190.47 μg/mL SPD1 while that of Captopril was 10 nM (868 ng/mL). Thin layer chromatography (TLC) also identified SPD1 as an ACE inhibitor. SPD1 acted as a mixed type inhibitor against ACE using FAPGG as a substrate. When 200 μg/mL SPD1 (10 μg) were added, Vmax and Km were 0.01 ΔA/min and 0.69 mM, respectively; without SPD, 0.03 ΔA/min and 0.42 mM. Trypsin was used for SPD1 hydrolysis and part of the reaction mixture was removed and analyzed at set times. ACE inhibitory activity increased from 52.47% to about 74.38% after 24 h hydrolysis. The results suggested that small peptides increased by trypsin hydrolysis of the SPD1 ACE inhibitory capacity also increased up to 24 h, then decreased, which may be due to the disappearance of some active ingredients. Six peptides, namely GFR, FK, IMVAEAR, GPCSR, CFCTKPC and MCESASSK, were synthesized based on the simulated trypsin digest of SPD1, then tested for ACE inhibitory activity. IC50 values of individual peptides were 94.25 ± 0.32, 265.43 ± 1.24, 84.12 ± 0.53, 61.67 ± 0.36, 1.31 ± 0.07 and 75.93 ± 0.64 μM, respectively, suggesting that CFCTKPC might represent the main domain for the ACE inhibition. The consumption of sweet potatoes may thus help alleviate hypertension and other diseases due to their SPD1 and hydrolysate content. [ABSTRACT FROM AUTHOR]

Recombinant thioredoxin h (Trx h2) overproduced in E. coli (M15) was purified by Ni2+chelate affinity chromatography as previously reported (Huang et al., 2004a). The molecular mass of Trx h2 was ca. 14 kDa as determined by SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide gel electrophoresis). Trx h2 had antioxidant (Huang et al., 2004b), dehydroascorbate reductase, and monodehydroascorbate reductase activities (Huang et al., 2008a). Trx h2 was shown by spectrophotometric methods to inhibit angiotensin converting enzyme (ACE) in a dose-dependent manner (50-200 μg/mL, with 31.9 ∼ 65.9% inhibition) using N-[3- (2-furyl) acryloyl]-Phe-Gly-Gly (FAPGG) as a substrate. A 50% inhibition (IC50) of ACE activity required 151.8 μg/mL of Trx h2 and 10 nM (868 ng/mL) of Captopril. TLC data also showed Trx h2 as an ACE inhibitor. Trx h2 acted as a mixed type inhibitor against ACE using FAPGG as a substrate. When 200 μg/mL Trx h2 were added, Vmax and Km were, respectively, 0.010 ΔA/min and 0.125 mM; without Trx h2 they were 0.0096 ΔA/min and 0.495 mM. Trypsin was used for Trx h2 hydrolysis over different time periods. ACE inhibitory activity was found to increase from 52% to about 72% after 16 h of hydrolysis. The results suggested that the ACE inhibitory capacity of small peptides increased through trypsin hydrolysis of Trx h2 up to 16 h and then decreased, which may have been due to the disappearance of some active ingredients. Four peptides, namely EVPK, VVGAK, FTDVDFIK and MMEPMVK, were synthesized based on the simulated trypsin digestion of Trx h2 and then tested for ACE inhibitory activity. The IC50 values of individual peptides were 1.73 ± 0.24, 1.14 ± 0.13, 0.42 ± 0.02, and 1.03 ± 0.58 mM, respectively, suggesting that FTDVDFIK might be the main active site of ACE inhibition. The results for Trx h2 and its hydrolysates might mean that consumption of sweet potato storage roots can aid in the control of hypertension and other diseases. [ABSTRACT FROM AUTHOR]

Soybean (Glycine max) is a major protein source for animal and human nutrition. The Bowman- Birk protease inhibitor (BBI), ranking 3rd of protein contents among soybean seed storage proteins, is a major antinutritional factor. BBI was incubated with 1 mM DTT at 37°C for 2 h and loaded directly onto a Sephadex G-25 gel column for purification. The molecular mass of the reduced form of BBI is ca. 8 kDa determined by SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide gel electrophoresis). The methodology we used includes total antioxidant status, (1,1-diphenyl-2-picryl hydrazyl) DPPH staining, DPPH radical scavenging activity, reducing power method, Fe2+-chelating ability, FTC (ferric thiocyanate) method, and protection calf thymus DNA against hydroxyl radical-induced damage. The oxidized and reduced form of BBI with a concentration of 200 μg/mL exhibited the highest activity (expressed as 4.74 ± 0.36 and 7.20 ± 0.20 mM Trolox equivalent antioxidative value, TEAC) in total antioxidant status test. In the DPPH staining the reduced form of BBI appeared as white spots when it was diluted to 12.5 μg/mL (a final amount of 0.6 μg). Like total antioxidant status, the reducing power, Fe2+-chelating ability, FTC activity and protection against hydroxyl radical-induced calf thymus DNA damage all showed that the reduced BBI exhibited higher antioxidative activities than the oxidized BBI. The results suggested that the reduced BBI exhibited higher antioxidative activities than the oxidized BBI in a series of in vitro tests. These findings provide one of the molecular bases for BBI applications to treat various serious diseases. [ABSTRACT FROM AUTHOR]