Botanical Studies (2011) 52: 15-22.
Sweet potato storage root thioredoxin h2 and their peptic hydrolysates exhibited angiotensin converting enzyme inhibitory activity in vitro
Guan-Jhong HUANG1,8, Hsien-Jung CHEN2,8, Kitanaka SUSUMU3, Jin-Bin WU4, Wen-Chi HOU5, Chieh-Hsi WU6, Ming-Jyh SHEU6, Shyh-Shyun HUANG2, and Yaw-Huei LIN7 *
1Institute of Chinese Pharmaceutical Sciences, China Medical University, Taichung 404, Taiwan
2Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung 804, Taiwan
3School of Pharmacy, College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi, Chiba 274-8555, Japan
4Graduate Institute of Pharmaceutical Chemistry, College of Pharmacy, China Medical University, Taichung, Taiwan
5Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan
6Department of Physiology, School of Medicine, China Medical University, Taichung 404, Taiwan
7Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei 115, Taiwan
(Received December 1, 2009; Accepted February 8, 2010)
ABSTRACT. Recombinant thioredoxin h (Trx h2) overproduced in E. coli (M15) was purified 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 (ig/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 [ig/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 [ig/mL Trx h2 were added, Vmax and Km were, respectively, 0.010 AA/min and 0.125 mM; without Trx h2 they were 0.0096 AA/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 士 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.
Keywords: Angiotensin converting enzyme (ace); Hydrolysis peptides; Sweet potato; Thioredoxin h2.
INTRODUCTION
Many bioactive peptides have common structural prop­erties that include a relatively short peptide residue length (e.g. 2-9 amino acids) and the possession of hydropho-bic amino acid residues in addition to proline, lysine, or arginine groups. Bioactive peptides are among the many functional components identified in foods. These are small protein fragments that have biological effects once they
are released during gastrointestinal digestion in the organ­ism or by previous in vitro protein hydrolysis. Bioactive peptides with immunostimulating (Fiat et al., 1993), anti-oxidant or angiotensin-converting enzyme inhibitor (Liu et al., 2007), antithrombotic (Scarborough et al., 1991), or bactericidal (Bellamy et al., 1993) functions have recently been the focus of research.
ACE (angiotensin converting enzyme, peptidyldipep-tide hydrolyase EC 3.4.15.1) is a glycoprotein and a dipep-tide-liberating exopeptidase classically associated with the renin-angiotensin system regulating peripheral blood pres­sure (Mullally et al., 1996; Lee et al., 2006). ACE removes a dipeptide from the C-terminus of angiotensin I to form
8These two authors contributed equally to this work. *Corresponding author: E-mail: boyhlin@gate.sinica.edu.tw; Fax: 886-2-2782-7954; Tel: 886-2-2789-9590 ext. 321.
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angiotensin II, a very hypertensive compound. Several endogenous peptides―such as enkephalins, p-endorphin, and substance P―were reported to be competitive sub­strates and inhibitors of ACE. Several food-derived pep­tides from casein (Maruyama et al., 1987), α-lactoalbumin, p-lactoglobulin (Pihlanto-Leppala et al., 1998), and mu­cilage (Huang et al., 2006) also inhibited ACE. Several antioxidant peptides (reduced glutathione and carnosine-related peptides) (Hou et al., 2003) and synthetic peptides also exhibited ACE inhibitor activities (Chen et al., 2003).
Thioredoxins, the ubiquitous small proteins with a redox active disulfide bridge, are important regulatory elements in a number of cellular processes (Besse and Buchanan, 1997). They all contain a unique active site, WCGPC, which is able to reduce disulfide bridges of tar­get proteins. Initially described as hydrogen carriers in ri-bonucleotide reduction in E. coli, they were found to serve as electron donors in a variety of cellular redox reactions (Holmgren, 1985). From genome sequencing data, a sig­nificant diversity of thioredoxin genes containing five dif­ferent multigenic families (f, m, x, o and h) was observed (Mestres-Ortega and Meyer, 1999; Balmer and Buchanan, 2002; Meyer et al., 2002). The ferredoxin-thioredoxin system (thioredoxins f and m) has been proven to regulate several enzymatic activities associated with photosynthetic CO2 assimilation in chloroplasts. Thioredoxin x contains a transit peptide similar to those required for chloroplast and mitochondria targeting; however, its function is not clearly defined (Mestres-Ortega and Meyer, 1999). A new type of plant mitochondrial thioredoxin, Trx o, was also shown to regulate the activities of several mitochondrial proteins by disulfide bond reduction (Laloi et al., 2001).
Thioredoxin h is generally assumed to be cytosolic, which was supported by the absence of a transit peptide in the genes cloned for the isoforms from Arabidopsis (Rivera-Madrid et al., 1995), Triticum aestivum (Gautier et al., 1998), tobacco (Marty and Meyer, 2001), germi­nating wheat seeds (Serrato et al, 2001), and barley seed proteome (Kenji et al., 2003). Moreover, the existence of several forms of thioredoxin h detected in wheat flour (Johnson et al., 1987), and spinach leaves (Florencio et al., 1988), supports the view that higher plants possess mul­tiple and divergent thioredoxin genes (Rivera-Madrid et al., 1995).
In our previous report, Trx h2 exhibited both dehydro-ascorbate reductase and monodehydroascorbate reductase activities (Huang et al., 2008a). Trx h2 also exhibited an-tioxidant activities against different radicals (Huang et al., 2004b). In this work we report for the first time that Trx h2 exhibited dose-dependent ACE inhibitory activity when Captopril was used as a positive control. Commercial bo-vine serum albumin (BSA), frequently found in the litera-ture as the peptide resource of ACE inhibitors, was chosen for comparison. The Ki values of Trx h2 against ACE were calculated. We also used trypsin to hydrolyze Trx h2 for different times, and the changes of ACE inhibitory activity were determined. The IC50 of ACE inhibitory activities by
synthetic peptides was also determined.
MATERIALS and METHODS
Materials
Tris, electrophoretic reagents, and silica gel 60 F254 were purchased from E. Merck Inc. (Darmstadt, Ger­many); Captopril was purchased from Calbiochem Co. (CA, USA); Seeblue prestamed markers for SDS-PAGE—including myosin (250 kDa), BSA (98 kDa), glutamate dehydrogenase (64 kDa), alcohol dehydrogenase (50 kDa), carbonic anhydrase (36 kDa), myoglobin (30 kDa), and lysozyme (16 kDa)─were from Invitrogen (Groningen, The Netherlands); FAPGG, ACE (1 unit, rabbit lung), coomassie brilliant blue G-250, peptides (GL Biochem, China), and other chemicals and reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
expression of thioredoxin h2 in E. coli
Thioredoxin h2 (Gene Bank accession number: AY344228; Trx h2) was expressed in E. coli. The cod­ing sequence was amplified from Trx h2 cDNA using an oligonucleotide (5'-GAG AGG ATC CAA TGG GAG GGG CT-3'), with a BamHI site (underlined) at the puta­tive initial Met residue, and an oligonucleotide (5'- ATT TGA AGC TTG ATT GAT GCT -3'), with a Hindlll site at the 3' end. The PCR fragment was subcloned in pGEM T-easy vector. The plasmid was then digested with BamHI and HindIll and subcloned in pQE-32 expression vector (QIAexpress expression system, Qiagen). The resulting plasmid, termed pQE-Trx h2, was introduced into E. coli (M15). Cultures of the transformed E. coli (M15) overex-pressed a protein of the expected molecular mass, which was purified by affinity chromatography in Ni-NTA col­umns (Qiagen) according to the method of Huang et al.
(2004b).
Protein staining of thioredoxin h2 on 15% denaturing polyacrylamide gels
Samples were mixed with sample buffer, namely 60 mM Tris-HCl buffer (pH 6.8) containing 2% SDS, 25% glycerol, and 0.1% bromophenol blue with or without 2-mercaptoethanol (2-ME). Coomassie brilliant blue G-250 was used for protein staining (Huang et al., 2004).
Protein stainings and electroblotting analysis of thioredoxin h2 in 15% SDS-PAGE gels
Trx h2 were examined by protein staining in 15% SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel elec-trophoresis) gels. Twenty microliter samples were mixed with 25 (iL sample buffer containing 60 mM Tris buffer
(pH 6.8), 2% SDS, 25% glycerol and 0.1% bromophenol
blue, with 2-ME in a final concentration of 14.4 mM, and heated at 100°C for 5 min for protein staining. Coomassie brilliant blue G-250 was used for protein staining. The Trx h2 was subjected to 15% SDS-PAGE according to the
HUANG et al. ― Thioredoxin h2 with angiotensin converting enzyme inhibitory activity
17
method of Huang et al. (Huang et al., 2007). After elec-trophoresis, gels were equilibrated in transfer buffer (25 mM Tris-HCl, pH 8.3, 150 mM glycine and 10 % (w/v) methanol). The separated proteins were transferred to an Immobilon PVDF membrane (Millipore, Bedford, MA) in transfer buffer at pH 8.3 for 1 h at 100 V Membranes were blocked for 2 h at room temperature in 5% nonfat dry milk powder and then incubated with polyclonal anti­bodies as the primary antibodies against thioredoxin from E. coli (Sigma, USA). After incubation, membranes were washed in Tris-buffered saline with 0.05% Tween (TBST) three times, 10 min each time, and incubated with anti-rabbit horseradish peroxidase-conjugated antibody. They were then washed in TBST three times, 10 min each time, and developed using diaminobenzidine (DAB) (Sigma,
USA).
Determination of the kinetic properties of ACE inhibition by thioredoxin h2
The kinetic properties of ACE (4 mU) without or with Trx h2 (200 (μg/mL) in a total volume of 250 μL were determined using different concentrations of FAPGG as
substrate (0.1 mM to 0.5 mM). The Km (without Trx h2)
and Km' (with Trx h2) were calculated from Lineweaver-Burk plots, where Km' was the Michaelis constant in the presence of 200 (g/mL Trx h2.
Determination of the ACE inhibitory activity of peptic hydrolysates of thioredoxin h2
Six mg of Trx h2 were dissolved in 1 mL of 0.1 M KCl buffer (pH 2.0). Then 0.1 mL of 12 mg of trypsin was added and hydrolysis was carried out at 37°C for 8, 12,
24, and 32 h. After hydrolysis, 0.5 mL of 0.5 M Tris-HCl
buffer (pH 8.3) was added, and the solution was heated at 100°C for 5 min to stop enzyme reaction. The trypsin was heated before thioredoxin h2 hydrolysis for the 0 h control reaction. Each of the 60 iL Trx h2 hydrolysates was used for determinations of ACE inhibition using spectropho-tometry.
Determination of ACE inhibitory activity by spectrophotometry
The ACE inhibitory activity was measured according to the method of Lee et al. (2003) with some modifica­tions. Four microliters (4 microunits) of commercial ACE was mixed with 50 μL of different amounts of Trx h2 or BSA (50, 100, and 200 (ig/mL), and then 200 μL of 0.5 mM N-[3-(2-furl) acryloyl]-Phe-Gly-Gly [FAPGG, dis­solved in 50 mM Tris-HCl buffer (pH 7.5) containing 0.3 M NaCl] was added. The decreased absorbance at 345 nm (ΔA inhibitor) was recorded over 5 min at room tempera­ture. Deionized water was used instead of sample solution for blank experiments (AA control). Captopril (molecular mass 217.3 kDa) was used as a positive control for ACE inhibitor (1.25, 2.5, 5, 10, 20, 40, and 80 nM). The ACE activity was expressed as AA 345 nm, and the ACE percent inhibition was calculated as follows: [1-(AA inhibitor/AA control)]x100. Means of triplicates were determined. The 50% inhibition (IC50) of ACE activity was defined as the concentrations of samples that inhibited 50% of ACE ac­tivity under experimental conditions.
Statistical analysis
Means of triplicates were calculated. Student's t test was used for comparison between two treatments. A dif­ference was considered to be statistically significant when p < 0.05.
RESULTS and DISCUSSION
Expression and purification of thioredoxin h2
To express sweet potato thioredoxin h2 in E. coli, the coding sequence of Trx2 was subcloned in a pQE-32 ex­pression vector so that sweet potato Trx h2 was produced with a 6x His-tag at the N-terminus. SDS-PAGE analysis of crude extracts from transformed E. coli (M15) showed a high level of a polypeptide with the expected molecular mass (ca. 14 kDa). This polypeptide was found as a sol­uble protein in the supernatant and was absent in protein extracts obtained from E. coli with pQE-32 vector. The ex­pressed protein was purified from crude extracts by Ni2+-chelate affinity chromatography, which yielded highly purified His-tagged thiredoxin h2. The next step of Trx h2 purification was the use of prepared SDS-PAGE (Figure 1). In our previous report, Trx h2 exhibited antioxidant, dehydroascorbate reductase and monodehydroascorbate reductase activities. The expression of Trx h2 with 6x His-tag protein did not affect the activity (Huang et al., 2008a; 2004b).
Determination of ACE inhibitory activity by TLC
The ACE inhibitory activity of Trx h2 was also deter­mined by the TLC method (Holmquist et al., 1979). The reactions between Trx h2 and ACE or BSA and ACE were measured according to the method of Anzenbacherova et al. (2001) with some modifications. Each 100 (μL of Trx h2 or BSA (225 μg/mL) was premixed with 15 microunits of ACE for 1 mm, and then 200 (μL of 0.5 mM FAPGG was added and allowed to react at room temperature for 10 min. Then 800 (iL of methanol was added to stop the reac國 tion. The blank experiment contained FAPGG only; in the control experiment, ACE reacted with FAPGG under the same conditions. Each was dried under reduced pressure and redissolved with 400 iL of methanol, and 50 μL was spotted on silica gel 60 F254. The FAPGG and FAP (ACE hydrolyzed product) were separated by TLC in 1-butanol-acetic acid-water, 4:1:1 (v/v/v), and observed under UV light.
Determination of ACE inhibitory activity of thioredoxin h2 by spectrophotometry
The purified Trx h2 was used for determinations of ACE inhibitory activity. Figure 2 shows the time course of the effect of different amounts of Trx h2 (0, 50, 100, and
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200 ( g/mL) on ACE activity (AA 345 nm). Compared with the ACE only (control), it was found that the higher the amount of Trx h2 added, the lower the AA 345 nm found during the 300-s reaction period. The results of Figure 2 show that purified Trx h2 could inhibit ACE activity in a dose-dependent manner.
Effects of thioredoxin h2, BSA and captopril on ACE activity shown by spectrophotometry
We also wanted to know whether BSA exhibited ACE inhibitory activity. Figure 3A shows the effects of Trx h2
(0, 50, 100, and 200 ( g/mL) and BSA (0, 50, 100, and 200 ( g/mL) or Captopril (Figure 3B; 0, 1.25, 2.5, 5, 10, 20, 40 and 80 nM; corresponding to 0, 108.5, 217, 434, 868, 1736, 3472 and 6944 ng/mL, respectively) on ACE
activity. BSA was found to show less ACE inhibitory ac­tivity (less than 15% inhibition) and no dose-dependent inhibition patterns. However, Trx h2 exhibited dose-dependent ACE inhibitory activity (50~200 (ig/mL, giv­ing, respectively, 31.9 ~ 65.9% inhibition). We calculated the 50% inhibition (IC50) of Trx h2 against ACE activity as 151.8 ( g/mL, compared to 10 nM (868 ng/mL) for Captopril, which was similar to what Pihlanto-Leppala et al. reported (7 nM). The IC50 of yam dioscorin was 250 (ig/mL (Hsu et al., 2002). Both BSA and purified Trx h2 were proteins, but only the purified Trx h2 showed specific dose-dependent ACE inhibitory activity. In the literature, the protein hydrolysates were used as sources to purify peptides as ACE inhibitors (Maruyama et al., 1987; Mul-
lally et al., 1996). We calculated the IC50 of Trx h2 against ACE activity as 151.8 (ig/mL, which was smaller than that of the synthetic peptide a-lactorphin (YGLF, 322.7 (g/ mL). Several identified peptide fragments exhibited much lower IC50 values than our purified Trx h2. For example, the Tyr-Pro of whey proteins were 8.1 (g/mL (Yamamoto et al., 1999), and the HHL of soybean proteins were 2.2
Figure 2. Inhibitory activity of thioredoxin h2 (0, 50, 100 and 200 ig/mL) of sweet potato storage root on ACE activity (AA 345 nm).
Figure 1. The protein staining (lane 1) and western blot analysis (lane 2) of thioredoxin h2. Both protein staining of Trx h2 with Coomassie brilliant blue R and Western blot analysis of sweet potato Trx h2 were performed on 15% SDS-PAGE gels. The gels were then transferred onto PVDF membranes that were probed with a 1:1000 (v/v) dilution of rabbit polyclonal antibod­ies raised against Trx h2, using goat-antirabbit horseradish per-oxidase assay as the second antibody. M indicates the Seeblue™ prestained markers of SDS-PAGE. Ten micrograms of Trx h2 were loaded in each well.
Figure 3. The effects of thioredoxin h2, albumin, and Captopril on ACE activity as determined by spectrophotometry. Thiore-doxin h2 (0, 50, 100 and 200 (ig/mL) or bovine serum albumin (0, 50, 100 and 200 ig/mL) was used. The inhibition of ACE (%) was calculated according to the equation [1-(AA inhibitor + AA control)] x 100%.
HUANG et al. ― Thioredoxin h2 with angiotensin converting enzyme inhibitory activity
19
(g/mL (Shin et al., 2001). On the other hand, several iden­tified peptide fragments exhibited much higher IC50 values than our purified Trx h2. Hydrolysates of whey proteins (a-lactalbumin and p-lactoglobulin), for example, had IC50 values of 345-1,733 (g/mL (Pihlanto-Leppala et al.,
2000); LAHKAL of a-lactalbumin hydrolysates, 406 (g/
mL; GLDIQK of p-lactoglobulin hydrolysates, 391 (g/
mL; and VAGTWY of p-lactoglobulin hydrolysates, 1,171
(g/ mL. In our previous paper, the IC50 of trypsin inhibitor from sweet potato was 188 ig/mL, higher than that of pu­rified Trx h2 (Huang et al., 2008b).
Determinations of ACE inhibitory activity of thioredoxin h2 by TLC
Figure 5. The Lineweaver-Burk plots of ACE (4 mU) without or with thioredoxin h2 (200 (g/mL) of sweet potato storage root using different concentrations of FAPGG (0.1 to 0.5 mM).
The FAPGG and FAP (product of ACE catalyzed hy­drolysis reaction) were separated by TLC using water saturated 1-butanol:acetic acid:water, 4:1:1 (V/V/V) as developing solvents according to the method of Holmquist et al. (1979). Figure 4 shows the qualitative results of the TLC chromatograms using silica gel 60 F254 which dem­onstrated the effects of 225 ig/mL of commercial BSA (lane 3) or Trx h2 (lane 4) on 15 microunits of ACE. Com­pared to the control test (lane 2), Trx h2 (lane 4) was found to better inhibit the ACE reaction, with less FAP produc­tion evident under UV light. However, the FAP amounts in the control test (lane 2) were similar to those found with BSA (lane 3). This demonstrated again that Trx h2 exhib­ited ACE inhibitory activity.
Determination of the kinetic properties of ACE inhibition by thioredoxin h2.
The Lineweaver-Burk plots of ACE (4 mU) without or with purified Trx h2 (200 (ig/mL) under different con­centrations of FAPGG are shown in Figure 5. The results indicated that purified Trx h2 acted as a mixed type inhibi­tor against ACE using FAPGG as a substrate. When 200 ig/mL Trx h2 were added, Vmax and Km were, respectively, 0.010 AA/min and 0.125 mM while without Trx hi they were 0.0096 AA/min and 0.495 mM. In conclusion, Trx h2 exhibited dose-dependent ACE inhibitory activity and act­ed as a mixed type inhibitor with respect to the substrate (FAPGG). A similar work calculated the Km as 0.255 mM FAPGG for ACE, and in the presence of purified dioscorin,
the calculated Km' was 0.3304 mM (Hsu et al., 2002).
Determination of the ACE inhibitory activity of peptic thioredoxin h2 hydrolysates
Trypsin has been frequently used for protein hydroly­sis to purify potential ACE inhibitory peptides (Pihlanto-Leppala et al., 2000). Therefore, we used it to hydrolyze Trx h2. Figure 6 shows the ACE inhibitory activity (AA 345 nm) of peptic Trx h2 hydrolysates. Figure 6 shows the ACE inhibition (percent) of peptic Trx h2 hydrolysates collected at different trypsin hydrolysis times. From the results, it was found that the ACE inhibitory activity in­creased from 52% (0 h) to about 72% (16 h). The ACE
inhibitor activities of peptic Trx h2 hydrolysates decreased after 16 h of hydrolysis (Figure 6) suggesting that some active ingredients got lost.
Kohmura et al. (1989) synthesized some peptide frag­ments of human p-casein and found that the length of those peptides had an influence on ACE inhibitory activ­ity. Namely, peptides composed of 3-10 amino acids with proline on the C-terminal were necessary as ACE inhibi­tors (Kohmura et al., 1990). Thus, the peptide Leu-Arg-Pro from food protein hydrolysates has been reported to be the most potent natural ACE inhibitor, with an IC50 value of 0.27 or 1.0 iM. Byun and Kim (2002) studied the ACE inhibitory activity of a series of dipeptides and indicated
Figure 4. The TLC chromatograms using silica gel 60 F254 showing the effects of thioredoxin h2 of sweet potato storage root or bovine serum albumin on ACE activity. Lane 1, blank test (FAPGG only); lane 2, control test (ACE reacted with FAPGG to produce FAP); lane 3, control test plus 225 (ig/mL bovine serum albumin; lane 4, control test plus 225 (g/mL thioredoxin h2. Each solution was dried under reduced pressure and redissolved with 400 (L methanol. Each 50 (L was spotted on silica gel 60 F254. The FAPGG and FAP were separated by water saturated 1-butanol:acetic acid:water, 4:1:1 (V/V/V). Arrows indicate the
positions of both FAP and FAPGG.
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In summary, Trx h2 exhibited dose-dependent ACE inhibitory activity. Trx h2 acted as a mixed type inhibi­tor toward ACE with an IC50 of 151.8 ig/mL. Its peptic hydrolysates also showed ACE inhibitory activities. Some peptides derived from food proteins were demonstrated to have antihypertensive activities against spontaneously hy­pertensive rats (Fujita et al., 2000; Yoshii et al., 2001). The potential for human hypertension control through the con­sumption of sweet potato deserves further investigation.
Acknowledgements. The authors want to thank the finan­cial support (CMU95-211) from China Medical University (CMU). This investigation was also supported by the "Ac­ademic Frontier (2007-2009)" and "High Tech Research Center (2007-2011)" projects for private universities, with a matching fund subsidy from MEXT (Ministry of Educa國 tion, Culture, Sports, Sciences and Technology of Japan).
Figure 6. ACE inhibitor activity of peptic hydrolysates of sweet potato thioredoxin h2. The plot shows the ACE inhibition (%) of peptic thioredoxin h2 hydrolysates obtained at different trypsin hy­drolysis times. The inhibition of ACE (%) was calculated according to the equation [1-(ΔA inhibitor + ΔA control)] x 100%.
that tryptophan, tyrosine, proline, or phenylalanine at the C-terminal and branched-chain aliphatic amino acids at the N-terminal were required for a peptide to bind to ACE
(Byun and Kim, 2002).
We measured the ACE inhibitory activity of some syn­thetic peptides based on Trx h2 gene sequences. Synthetic peptides were designed by simulating the trypsin cutting sites of sweet potato Trx h2 gene (accession number: AY344228) products (pH>2, http://expasy.nhri.org.tw/ tools/peptidecutter/). Four new inhibitory peptides (Table 1) for ACE, namely EVPK, VVGAK, FTDVDFIK, and MMEPMVK, were synthesized according to simulation. 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. These results demonstrated that simulated synthetic peptides from peptic Trx h2 hydrolysates exhibited ACE inhibitory activities. Our work suggests that (1) FTDVDFIK might represent the main active site for the ACE inhibition; (2) peptides with antihypertensive, immunomodulatory, and antioxidant activities have marked structural similarities, and these may be used as criteria for selecting or design-ing the multifunctional ingredients of functional foods to control cardiovascular diseases.
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Table 1. Thioredoxin h2 peptides with ACE inhibitor activity.
TRX h2 peptides
I
C50
EVPK
1.73
±
0.24
mM
VVGAK
1.14
±
0.13
mM
FTDVDFIK
0.42
±
0.02
mM
MMEPMVK
1.03
±
0.58
mM
Note: The sequence of Trx h2 had pre-pro-sequence. These sequences were retrieved from the NCBI (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov) with accession number AY344228.
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21
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甘藷塊根中硫氧化還原蛋白h2及其合成之胜肽具有血管收縮
素轉化酶抑制活性
黃冠中1 陳顯榮2 北中進3 吳金濱4 侯文琪5
吳介信6 許明志6 黃世勳2 林耀輝7
1中國醫藥大學中國藥學研究所
2中山大學生命科學系
3曰本大學藥學系
4中國醫藥大學藥物化學研究所
5臺北醫學大學生藥學研究所
6中國醫藥大學藥學系
7中央研究院植物暨微生物研究所
在大腸桿菌（M15)中大量表現重組蛋白質硫氧化還原蛋白h2 (Trx h2)，利用鎳離子螯合之親和性管
柱純化。Trx h2經SDS-PAGE分析其分子量約為14 kDa 。由於Trx h2具有抗氧化活性，去氫抗壞血酸
還原酶'單去氫抗壞血酸還原酶的活性。Trx h2以(N-(3-[2-furyl] acryloyl)-Phe-Gly-Gly) (FAPGG)為受
質，利用分光光度計的方法分析抑制血管收縮素轉化酶(angiotensin converting enzyme, ACE)的能力，其
效果隨劑量增加而增加（50到200 ig/mL TI ，分別抑制31.9-65.9 %血管收縮素轉化酶活性）。Trx h2對
於血管收縮素轉化酶之50%抑制濃度(IC50)為151.8 ig/mL '對照組Captopril為10 nM (868 ng/mL)。另
外利用螢光silica TLC偵測FAPGG及其水解產物FAP ，結果也顯示Trx h2對於血管收縮素轉化酶有抑
制的效果。Trx h2對於血管收縮素轉化酶是屬於混合型抑制。而當加入200 ig/mL Trx h2時，Vmax和Km
分別為0.010 AA/min和0.125 mM ；而未入Trx h2時'Vmax和Km分別為0.0096 ΔA/min和0.495 mM利
用胰蛋白酶以不同時間水解Trx h2時，發現反應16小時時其血管收縮素轉化酶活性有抑制的效果可以
從52 % (0 h)增加到72 % (16 h)。由結果可知小分子的胜肽會隨著水解時間增加且血管收縮素轉化酶活
性抑制也有增加，但水解時間超過16 h時，血管收縮素轉化酶活性抑制會降低，可能是由於一些胜肽
的結構被破壞。利用電腦模擬胰蛋白酶水解Trx h2的結果，四種人工合成具有抑制血管收縮素轉化酶活
性胜肽：EVPK, VVGAK, FTDVDFIK和MMEPMVK '測定其IC50為1.73 ± 0.24, 1.14 ± 0.13, 0.42 ± 0.02
和1.03 ± 0.58 mM 。結果發現FTDVDFIK具有很好的抑制血管收縮素轉化酶活性。當人們食用甘藷塊根
時，Trx h2及其胜肽也許對於高血壓和其他疾病的控制是有益的。
關鍵詞：甘藷；血管收縮素轉化酶；硫氧化還原蛋白h2 ；水解胜肽。