Isolation, Purification and Molecular Weight Determination of Antihypertensive Peptides Derived from

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1 2011, Vol. 32, No ,2 1 1, * 1 1 ( ) AS.1398 (Porphyra haitanesis)ace Sephadex G-15 (RP-HPLC) (MALDI-TOF-MS) 2000D ACE IC mg/mL Sephadex G-15 6 E IC mg/mL E RP-HPLC 6 ACE 89.54% 6 RP-HPLC Tyr Val Phe MALDI-TOF-MS 8 m/z m/z D 3 Val 2 Phe 1 Tyr N Val 18 A C E Isolation, Purification and Molecular Weight Determination of Antihypertensive Peptides Derived from Porphyra haitanesis LIU Shu-ji 1,2 WANG Yin 1 WU Cheng-ye 1, * SU Yong-chang 1 LIU Zhi-yu 1 (1. Fisheries Research Institute of Fujian, Xiamen , China 2. College of Food Science, Fujian Agriculture and Forestry University, Fuzhou , China) Abstract Porphyra haitanesis was hydrolyzed with AS.1398 neutral protease, and the hydrolysate was filtrated sequentially through ultra-filtration membranes with molecular weight cutoff (MWCO) 2000 D and 8000 D to obtain the fraction with the highest ACE-inhibitory activity, of which the molecular weight was below 2000 D. The fraction was then purified by Sephadex G-15 gel permeation chromatography into 6 peaks, in which peak E had the strongest ACE inhibitory activity with an IC50 of 0.67 mg/ml, and peak E was further fractionated by RP-HPLC to obtain peak No.6 with the highest ACE-inhibitory activity, reaching up to 89.54%. In the end, peak No.6 was injected into RP-HPLC system once again, and two protein peaks were observed in the chromatogram, suggesting that peak No.6 is not a single component and is still in need of further purification. The amino acid composition of peak No.6 was mainly composed of Tyr, Val and Phe. Meanwhile, the MALDI-TOF-MS spectrum of peak No.6 displayed 8 major proton peaks, the peak with the strongest signal intensity was m/z , and 4 of them were fluctuated around m/z 860, indicating that the mean molecular weight of peak No.6 is approximately 860 D. We speculated that antihypertensive peptides derived from Porphyra haitanensis consisted of 3 Val residues, 2 Phe residues and 1 Tyr residue, of which at the N-terminal Val was located, and that the number of possible ranking sequences was 18. Key words Porphyra haitanensis antihypertensive peptide isolation purification molecular weight S985.4 A (2011) (2008N0202) (1981 ) cute506636@163.com * (1953 ) wcy@fjscs.ac.cn

2 , Vol. 32, No. 02 (Porphyra haitanesis) 1:2 0(g/m L) 7 % 30% h min [ 1 ] 8000r/min 15min 10 m [2] 8000D 2000D 3 (M 8000D) (2000D M 8000D) ( M 2000 D ) A C E (angiotensin converting enzyme inhibitory Sephadex G-15 peptides ACEIPs) 0.5g/mL 0.5mL 2mL/min 280nm [3] 1979 Oshima [4] A C E 6 ACE 1500D Nakamura [5] (RP-HPLC) ( SH R ) 20% B ( 0.1% TFA ) Saito [6] 9 ACE (Microsorb-MV C18) 50mg/mL A ( ACE [7-8] [ 9] [1 0] [1 1] [12] [13] [ 14 ] [ 15 ] [ 16 ] [ 17 ] AS MALDI-TOF-MS( [ 1 8 ] Sephadex G-15 -CH-4-OH- (HCCA) (RP-HPLC) TA(0.1% TFA : =2:1 V/V) [19] (Hip-His-Leu HHL) Sigma Sephadex G-15 ( ) TFA(TEDIA) Pharmacia Tedia (ACE) ACE Prostar 240 Varian % TFA ) 20 L 5% 30% B 40min 0.5mL/min 220nm ) 0.1mol/L 5 L 5 L 1 L Reflex MALDI-TOF 20kV 16.3kV 23kV N2 337nm Pa D Mark Cushman 2mL 50 L 200 L 05mol/L Reflex MALDI-TOF Bruker HD- HHL( 0.3mol/L NaCl ph8.3) 37 1 SBS-100 5min 50 L 0.4U/mL ACE LZB min 0.2mL 1mol/L LGJ-2 1.2mL R r/min 5min 0.8mL SP ( 1h) 4mL 2min 228nm 1.2 A 0.2mL 1mol/L 250g AS.1398

3 2011, Vol. 32, No A B C D E F Aa Ab ACE /%= 100 G H I( 1) A CE IC 50 2 Aa E IC mg/mL ACE Aa Ab G IC mg/mL (IC 50) 50% Sephadex G D /(mg/ml) IC50 E 2.3 RP-HPLC SPSS (Probit) E RP-HPLC I C ACE 89.54% % RP-HPLC AS.1398 E 6 RP-HPLC 8000D 2000D 3 2 ( 3) RP-HPLC ACE IC IC 50 IC mg/mL IC 50 1/2 2000D 1 Table 1 AC-inhibitory IC50 of hydrolyzed Porphyra haitanesis and its ultra-filtration fractions (M 8000D) (2000D M 8000D) (M 2000D) IC50/(mg/mL) ( 2) ACE 3 3 AU E RP-HPLC Fig.2 RP-HPLC chromatogram of Sephadex G-15 separated peak E /min A280nm A C B D E F G H I /h 1 Sephadex G-15 Fig.1 Sephadex G-15 separation profile 2 Sephadex G-15 ACE of peak E Table 2 AC-inhibitory IC50 of Sephadex G-15 separated peaks A B C D E F G H I /% IC50/(mg/mL) g/mL Sephadex G-15 AU /min 3 6 RP-HPLC Fig.3 Second RP-HPLC chromatogram of RP-HPLC separated peak No.6 3 RP-HPLC E ACE Table 3 ACE inhibition rate of various RP-HPLC separated fractions 2.4 HPLC 6

4 , Vol. 32, No Table 4 Amino acid profile of peak No. 6 Asp Thr Ser Glu Gly Ala Cys Met Ile Leu Tyr Phe Lys Arg Pro Val /(mg/100ml) /(mg/100ml) mg/100mL (Tyr) (Phe) (Val) 80.6% 6 MALDI-TOF-MS 860D = m/z ( ) n 18 (n 1) (n ) ( 4 ) 8 4 n= Val(V) 4 m/z D 3 Val 2 Phe 1 Tyr Cheung Sephadex G-15 (RP-HPLC) 6 [20] 1000D A C E % 1000D 860D 3 V a l 2 Ph e 1 Ty r N ACE IC mg/mL Val 2000D 8000D 18 ACE ACE 2000D IC mg/mL IC mg/mL Sephadex G-15 E IC mg/mL A A D I RP- HPLC ACE IC50 ACE 6 ACE 89.54% RP- HPLC 2 RP-HPLC 1 RP-HPLC 6 Tyr Val Phe m/z 860 m/z Tyr (T) Phe (P) m/z MALDI-TOF-MS Fig.4 MALDI-TOF/MS spectrum of peak No V T P= V+P= [21] C ( Trp Tyr Phe) Pro ACE N ( Val Leu Ile) ACE [2 2] N Val C Tyr Phe N Val 5 C 4 2 C 3 1 C 2 2 =18 3 AS.1398 ACE [1]. [J]., 1999, 18(4): [2],,. [J]., 2005, 27(5): [3],,,. [J]., 2004, 25(5): 3-6. [4] OSHIMA G, SHIMABUKURO H, NAGASAWA K, et al. Peptide inhibitors of angiotensin -converting enzyme in digest of gelatin by bacterial collagenase[j]. Biochim Biophys Acta, 1979, 566(1): [5] NAKAMURA Y, MASUDA O, TAKANO T, et al. Decrease of tissue angiotensin -converting enzyme activity upon feeding sour milk in spontaneously hypertensive rats[j]. J Biosci Biotech Biochem, 1996, 60

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