Preparation Of Angiotensin I-converting Enzyme And Isolation Of ACE Inhibitory Peptides From Abalone(Haliotis Discus Hannai)

Hypertension,one of the common chronic diseases,is mainly regulated by angiotensin I-converting enzyme and aminopeptidases.Angiotensin I-converting enzyme(ACE,EC 3.4.15.1)is a kind of membrane bound carboxyl peptidase found predominantly in mammal lungs.It generates the vasopressor angiotensin ?(Ang ?)from angiotensin I(Ang I)and also inactivates the potent vasodilator bradykinin,thus leading to a rise in blood pressure.On the other hand,aminopeptidase B(APB,EC 3.4.11.6)is involved in the generation of vasodilator AngIV and bradykinin,which plays an important role in lowering blood pressure.Thus,APB and ACE work together to keep blood pressure homeostasis.As a basic model for disease research,pigs are often used in the study of hypertension.In this study,tool ACE was prepared and purified from fresh pig lungs by a new method combining sodium chloride salting and column chromatography.Results showed that the enzyme activity of the prepared ACE was comparable to that of ACE derived from rabbit lung purchased from sigma and was stable in the condition of frozen storage which could be maintained for more than one year.Further study showed the molecular weight and pI value of the purified ACE was approximately 180 ku and 5.7,respectively.By MALDI-TOF/TOF mass spectrometry analysis,17 peptide fragments with 243 amino acid residues were obtained and the sequences of these peptide fragments were identical to an angiotensin I-converting enzyme from Sus scrofa.Enzymatic properties analysis showed ACE revealed an optimum temperature of 40°C and an optimum pH of 8.At the same time,APB was purified from pig muscle.It displayed a single band in SDS-PAGE,and its apparent molecular weight was about 100 ku.The optimal synthesis condition of APB is at 35°C,pH 7.The kinetic experiments showed the Km value of APB was 1.5 ?mol/L,kcat value was 47.9 S-1 and catalytic efficiency(kcat/Km)was 31.9(?mol/L)-1 S-1.In a word,the preparation,purification and characterization of ACE and APB indeed laid the foundation for screening hypotensive substances.On these bases,ACE inhibitory peptides were prepared from abalone(Haliotis discus discus)muscle.The hydrolysates of abalone with ACE inhibition rate of 72% were obtained by one step enzymatic hydrolysis using compound protease.Response surface analysis showed the optimum hydrolysis condition was at 50°C,pH 7.0,with enzyme dosage of 0.3% and incubation for 1.7 h.The abalone polypeptide was then obtained by a 3 ku ultrafiltration membrane and it revealed good thermal stability,pH stability and digestion stability.In addition,the 3 ku fraction was then assessed for antihypertensive activity in vivo using spontaneously hypertensive rats(SHRs)and a drop of 42 mmHg in SBP was observed 8 h after oral administration at a dose of 200 mg/kg body weight.At the same time,we found the decrease in blood pressure was accompanied by an increase in insulin-regulated aminopeptidase(IRAP,EC 3.4.11.3)activity and a decrease in APB and aminopeptidase N(APN,EC 3.4.11.2)activity in plasma.In order to identify the efficient ACE inhibitory peptide,abalone polypeptide was then purified by chromatographies including Sephadex G-15 and reversed-phase high performance liquid chromatogram(RP-HPLC).After identification and synthesis,IACG peptide exhibiting corresponding ACE inhibitory activity with IC50 value of 70.5 ?g/mL was obtained.In summary,successful preparation of abalone derived ACE inhibitory peptides not only enriched the nutrition and health care of abalone but also could help to elucidate the antihypertensive mechanism of ACE inhibitory peptides.