Sweet potato storage root thioredoxin h2 and their peptic hydrolysates exhibited angiotensin converting enzyme inhibitory activity in vitro

Recombinant thioredoxin h (Trx h2) overproduced in E. coli (M15) was purifed by Ni²⁺-che-late 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, V_max and K_m 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.