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

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2011, Vol. 32, No. 02 213 1,2 1 1, * 1 1 (1. 361012 2. 350002) AS.1398 (Porphyra haitanesis)ace Sephadex G-15 (RP-HPLC) (MALDI-TOF-MS) 2000D ACE IC50 0.73mg/mL Sephadex G-15 6 E IC50 0.67mg/mL E RP-HPLC 6 ACE 89.54% 6 RP-HPLC 2 6 6 Tyr Val Phe MALDI-TOF-MS 8 m/z 861.17 4 m/z 860 6 860D 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 361012, China 2. College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002, 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 861.17, 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 1002-6630(2011)02-0213-05 2010-03-17 (2008N0202) (1981 ) E-mail cute506636@163.com * (1953 ) E-mail wcy@fjscs.ac.cn

214 2011, Vol. 32, No. 02 (Porphyra haitanesis) 1:2 0(g/m L) 7 % 30% 50 10 12h 100 15min [ 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 1.2.2 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] 2 1.2.3 (RP-HPLC) ( SH R ) 20% B ( 0.1% TFA ) Saito [6] 9 ACE (Microsorb-MV 100-5 C18) 50mg/mL A ( ACE [7-8] [ 9] [1 0] [1 1] [12] [13] [ 14 ] [ 15 ] [ 16 ] [ 17 ] AS.1398 1.2.4 MALDI-TOF-MS( [ 1 8 ] Sephadex G-15 -CH-4-OH- (HCCA) (RP-HPLC) TA(0.1% TFA : =2:1 V/V) [19] 1 1.1 (Hip-His-Leu HHL) Sigma Sephadex G-15 ( ) TFA(TEDIA) Pharmacia Tedia (ACE) 1.2.5 ACE Prostar 240 Varian 1.2.1 0.1% 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 1.2 10-5 Pa 500 1500D 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-4 37 30min 0.2mL 1mol/L LGJ-2 1.2mL R205 3500r/min 5min 0.8mL SP-75 85 ( 1h) 4mL 2min 228nm 1.2 A 0.2mL 1mol/L 250g AS.1398

2011, Vol. 32, No. 02 215 9 A B C D E F Aa Ab ACE /%= 100 G H I( 1) A CE IC 50 2 Aa E IC50 0.67mg/mL ACE Aa Ab G IC50 0.93mg/mL (IC 50) 50% Sephadex G-15 2000D /(mg/ml) IC50 E 2.3 RP-HPLC SPSS (Probit) E RP-HPLC 8 1 2 3 4 5 6 7 I C 5 0 2 6 ACE 89.54% 2.1 5 7 8 60% RP-HPLC AS.1398 E 6 RP-HPLC 8000D 2000D 3 2 ( 3) RP-HPLC ACE IC50 1 1 IC 50 IC50 0.73mg/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) 1.27 2.02 1.09 0.73 8( 2) ACE 3 3 AU 2.0 1.5 1.0 0.5 1 2 3 2 E RP-HPLC Fig.2 RP-HPLC chromatogram of Sephadex G-15 separated peak E 4 5 10 20 30 40 /min 6 7 8 2.2 0.5 A280nm 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 A C B D E F G H I 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 1 /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 2 1 2 3 4 5 6 7 8 1 A B C D E F G H I /% 6.84 6.51 29 31.33 72.51 89.54 65.16 61.60 IC50/(mg/mL) 9.61 7.46 6.63 4.49 0.67 1.37 0.93 5.34 13.65 0.5g/mL Sephadex G-15 AU 0.4 0.3 0.2 0.1 0 5 10 15 20 25 30 35 40 45 50 /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

216 2011, Vol. 32, No. 02 4 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) 1.27 0.90 1.35 1.15 2.06 2.07 1.56 2 0.45 0.59 25.32 14.41 0.24 0.49 0.66 13.55 /(mg/100ml) 66.09 4 66.09mg/100mL (Tyr) (Phe) (Val) 80.6% 6 MALDI-TOF-MS 860D = m/z (600 1200) n 18 (n 1) (n ) ( 4 ) 8 4 n=6.16 3 Val(V) 4 m/z 694.03 747.30 952.25 1078.05 6 8 860D 3 Val 2 Phe 1 Tyr Cheung Sephadex G-15 (RP-HPLC) 6 [20] 1000D A C E 8 9. 5 4 % 1000D 860D 3 V a l 2 Ph e 1 Ty r N ACE IC 50 0.4mg/mL Val 2000D 8000D 18 ACE ACE 2000D IC 50 0.73mg/mL IC 50 1.27mg/mL Sephadex G-15 E IC50 0.67mg/mL A A 1 5 0 0 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 861.17 117. 1 5 Tyr (T) 181. 19 Phe (P) 165. 19 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 694.0346 747.2993 845.1714 861.1667 867.0764 877.1038 952.2458 m/z 1078.0506 700 800 900 1000 1100 1200 4 6 MALDI-TOF-MS Fig.4 MALDI-TOF/MS spectrum of peak No. 6 860 V T P=396.47 2V+P=399.49 [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): 465-467. [2],,. [J]., 2005, 27(5): 56-62. [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): 128-137. [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

2011, Vol. 32, No. 02 217 (3): 488-489. [6] SAITO Y, WANEZAKI K, KAWATO A, et al. Structure and activity of [J]., 1998, 23(2): 49-51. [9],,. [J]., 2006, 27(5): 117-119. [10] FUJITA H, SASAKI R, YOSHIKAWA M, et al. Potentiation of the antihypertensive activity of orally, a vasorelaxing peptide derived from ovalbumin, by emulsification in egg hosphatidylchoine[j]. J Biosci [19],,,. - Biotech Biochem, 1995, 59(12): 2344-2345. [J]., 2001, 19(3): 222-225. [11],,,. [J]., 2003, 24(10): 120-123. [12] UKEDA H, MATSUDA H, OSAJIMA K, et al. Peptides from peptic hydrolyzate of heated sardine meat that inhibit angiontensin -converting enzyme[j]. Nippon Nogeikagaku Kaishii, 1992, 66(1): 25-29. [13] FUJITA H, YOKOSHIKAWA M, LKPNM: A prodrug type ACE inhibitory peptide derived from fish proteins[j]. Immunopharmacology, 1999, 44(1/2): 123-127. [14]. [D]. :, 2007. agiotensin -converting enzyme of peptides originating from sake and sake lees[j]. Biosci Biotech Biochem, 1994, 58(10): 1767-1771. [15] SUETAUNA K. Isolation and characterization of angiotensin -converting enzyme inhibitor dipeptides derived from Allium sativum L. (garlic) [7]. [D]. :, [J]. J Nutr Biochem, 1998, 9(7): 415-419. 2006. [16],,,. [J]. [8],. ( ): ACEI, 2007, 32(7): 28-31. [17] OKAMOTO A, HARAGATA H, MATSUMOTO E, et al. Agiotensin converting enzyme inhibiroty activities of various fermented foods[j]. Biosci Biotech Biochem, 1995, 59(6): 1147-1149. [18],,,. [J]., 2008, 12(4): 64-68. [20],,,. [J]., 2007, 32(7): 28-31. [21] CHEUNG H S. Spectrophotometric assay and properties of the angiotensin I-converting enzyme of rabbit lung[j]. Biochem Pharmacol, 1971, 20(7): 1637-1648. [22],,,. [J]., 2005, 4(1): 46-50.

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