A Ne w Method of Separation and Bioactivity Assay of Flammulin

ISSN 100727626 CN 1123870ΠQ 2003 4 Chinese Journal of Biochemistry and Molecular Biology 19 (2) :234 239,,,,, (, 250100) 3, :, DEAE2Sephadex A250, CM2Sephadex C250,, 23 700, pi 819, max 276 278 nm, min 250 nm,, S 180,,,, Q51 A Ne w Method of Separation and Bioactivity Assay of Flammulin ZHOU Kai2song, PENGJun2feng, CHANG Ning, ZHANG Han2xing, GONG Feng, ZHANG Chang2kai 3 ( National Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China) Abstract Flammulin, an anti2tumor protein, was purified from the aqueous extract of basidiomes of Flammulina velutipes The purification process included ammonium sulfate and ethanol fractionation, DEAE2 Sephadex A250 and CM2Sephadex C250 ion2exchange chromatography Flammulin was purified to electrophoretic homogeneity and crystallized by microdialysis With a molecular mass of 23 700, pi 819, max = 276 278 nm, min = 250 nm, flammulin was characterized by its lack of Met The product was found to have marked anti2tumor effect on Sarcoma 180 and Ehrlich ascites tumor No toxicity has been detected Key words flammulin, ion exchange chromatography, bioactivity assay ;,, 20 2 N Komatsu [1, 2 ] g, ; SDS2PAGE, pi Pharmacia ;, Merck ; N Komatsu [3 ] 112, Watanabe [2 ], 2 kg, 015 %, S 180 1 111 :2002208208, :2002210214 (No 2000042212) 3 Tel : (0531) 8364427,E2mail :ckzhang @life sdu edu cn,,1972 2, Received :August 8,2002 ;Accepted :October 14,2002 Supported by Ministry of Education of China for Doctor Foundation ( No 2000042212) 3 Corresponding author Tel : (0531) 8364427 E2mail :ckzhang @life sdu edu cn

2 : 235,, 66 %, :190 300 nm, 50 % : 1 mg, (VΠV), - 20, 6 molπl HCl 110 24 h, 75 % (VΠV), - 20, (OPA), LC24A 0102 molπl Na 2 HPO 4 2NaH 2 PO 4 (ph616) ;, DEAE2Sephadex A250, : 5 molπl NaOH, 110 24 h,,,, 01005 molπl NaAc2HAc (ph 516) 116, CM2Sephadex C250, : 60,, NaAc2HAc 012 ml (1 10 7 Πml) (010052011 molπl), 6, 10 113 015 mlπ ;, Watanabe DEAE2Sephadex2 A250, CM2Sephadex2C250, 60 d, 0102 molπl Na 2 HPO 4 2NaH 2 PO 4 (ph718), = [ ( T - C) 100 %]ΠC, NaCl (0 110 molπl), C ; T 14 mlπh, 214 ml [4 ] S 180 Lowry [5 ], (BSA) :, 012 ml 116 (2 10 7 Πml) S 180 ( ) 114,S 180 ( ) 6, 10 SDS2PAGE Laemmli [6 ], : 015mlΠ ; 12 %, 5 %, 20 ma, R250, 1125 mgπkg 215 mgπkg 5 mgπkg 10 mgπkgπ, 10 d, ( IEF) Chiari [7 ],,, (315 10) 715 %, 1 molπl, 1 molπl NaOH1 R250 (CZE) Wheat [8 ],PΠACETMMDQ,BECKMAN ;, 30 cm, 5 cm, : 20, 25 :012 molπl 30 psi 2 min ; 30 psi 1 min ; 2 30 psi 2 min ;015 psi 5 s ;20 kv 5 8 min ;PDA 258 nm Molisch [9 ], 1 ml, 7 14 d,, 1 Molisch (5 2 100 ml 95 % ),, 1 ml,, 1125 mgπkg 215 mgπkg 5 mgπkg 10 mgπkg, 10 d, = [ ( T - C) 100 %]ΠC T ; C :, mgπ 10,, 117 : 30 ( ), 5, 6,, 2, 10 gπm1 211, CM2Sephadex2C250 Fig1 1, 115 UV2240 Fig 2 4

236 19 Fig 1 Improved Ion2exchange column chromatography purification of flammulin The active fractions from DEAE2Sephadex2A250 column chromatography were applied to a CM2Sephadex2C250 column pre2 equilibrated with 0 02 molπl Na 2 HPO 4 2NaH 2 PO 4 ph718 After the unbound materials were eluted, the column was eluted with a linear gradient of 0 1 molπl NaCl in 0102 molπl Na 2 HPO 4 2NaH 2 PO 4, ph 718 The activities of the fractions were determined as described under Materials and Methods Fig 3 SDSΠPAGE analysis of flammulin purified by improved anion2exchange column chromatography 1 Molecular mass markers from Pharmacia 2 Flammulin Fig 4 IEF patterns of Flammulin 1 Molecular mass markers from Pharmacia 2 Flammulin Fig 2 SDS2PAGE analysis of flammulin purified by the method of Watanabe 1 Molecular mass markers from Pharmacia 2 Active fractions Fig 2, Watanabe, 2 ( Fig 3 5) 23 700, pi 819 (Fig 5), Fig 5 Capillary zone electrophoretogram for flammulin Running voltage 25 kv, Borax2NaOH buffer (ph 9 4)

2 : 237 Molisch, 21212 Table 1 Gilliland [4 ] Table 1 Amino acid composition of flammulin, Fig 6 Fig 6 Crystals of flammulin grown in polyethylene glycol Maximum dimension is 4 mm conditions that cause crystals to crack and melt Changed from the crystallization 2 2 2 2 1 Fig 7 max 276 278 nm, min 250 nm, Ala (35) Met Amino acid Residues in flammulin molecule Ala 35(34179) Arg 15(14153) NH 3 25(25102) Asp 33(33125) Cys 7(7106) Val 6(5177) Glu 17(16169) Gly 24(24118) His 4(3196) Ile 5(4188) Leu 14(14123) Lys 3(3103) Met 0(0100) Phe 8(7186) Pro 19(18179) Ser 14(14100) Thr 19(19112) Trp 1(1102) Tyr 11(10169) Total residues 235 Fig 7 UV2absorption spectrum of flammulin 213 21311 215 mgπkg 5 mgπkg 10 mgπkg ( Table 2) 21312 S 180 S 180 5 mgπkg, 55117 %, 215 mgπ kg 5 mgπkg 10 mgπkg ( Table 3 Table 4) Table 2 Effect of flammulin on the span of lives of mice injected with Ehrlich ascites tumor Group flammulin, Πmg kg - 1, 10 Numbers of mice BeginΠEnd 21313, 10 mgπkg 57143 11174 84142 117130 ( P < 0101) ( Table 5) Average survival Control 1 10Π0 20 00 days Ratio of the survival period prolongation( %) Value of P 1 1125 10Π3 31 40 61 56 < 0 05 2 215 10Π4 36 25 86 52 < 0 01 Control 2 10Π0 18 87 3 5 10Π3 37 50 92 95 < 0 01 4 10 10Π3 38 36 98 77 < 0 02

238 19 Table 3 Effect of flammulin on tumor S 180 in mice Group flammulin, Πmg kg - 1 10 miceπg solid tumor in mice Πg Control 4161 1145 0187 Tumor2 inhibiting ratio ( %) Value of P 1 215 6105 1104 0166 28127 0105 2 5 3120 0165 0142 55117 0102 Table 4 Effect of flammulin on Ehrlich ascites tumor in mice Group flammulin, Πmg kg - 1 miceπg, 10 solid tumor in miceπg Control 1 7125 1105 0142 Tumor2 inhibiting ratio ( %) Value of P 1 1125 4113 0169 0139 34128 0 05 2 215 3138 0157 0131 45171 0 01 Control 2 9163 1103 0145 3 5 5125 0153 0125 48154 0 01 4 10 1178 0156 0126 45163 0 02 Table 5 Effect of immune apparatus in mice treated with flammulin Group flammulin thymus solid spleen Πmg kg - 1, 10 Πmg Πmg Control 1 25169 3164 60182 17173 1 1 25 29185 4101 69183 1112 2 2 5 27167 2198 68104 13199 Control 2 18198 4143 57143 11174 3 5 20102 4106 61102 18126 4 10 20113 3176 84142 17130 3 P < 0101,compared with control group 21314 Table 6 LD 50 = 98136 mgπkg, LD 50 = 93136 mgπkg, E max = 01000244, 95 % = 70179 136168 mgπkg, 95 % = 32195 mgπkg Table 6 Median lethan dose (LD 50 ) of flammulin on mice (Bliss s method) flammulin Πmg kg - 1 flammulin log Number of mice Number of death Death ratio ( %) Factual probability unit Final probability unit Regress probability 200 21301 6 6 100 6173 710285 710287-010002 100 21000 6 3 50 5103 510472 510473-010001 80 11900 6 1 16 7 4103 414092 414094-0 0000 60 11778 6 1 16 7 4103 315871 315870 010000 40 11602 6 0 0 3127 214281 214280 010001 unit Error : 200 mgπkg 100 mgπ kg 80 mgπkg 20 min,,,,24 h 1 2 g,, 24 36 h, 48 h 60, mgπk 40 mgπkg 1 h,,4 h : 200 mgπkg (24 h), 48 h 100 mgπkg, 3,,, ( References) 1 Komatsu N, Terakawa H, Nakanishi K Flammulin, a basic protein of Flammulina velutipes with anti2tumor activities J Antibiotics, Ser A, 1963,16(3) :139 143 2 Watanabe Y, Nakanishi K, Komatsu N Flammulin, an anti2tumor substance Bull Chem Soc, Japan, 1964,37(5) :747 750 3,, ( Feng Yong2jun, Zhang Chang2kai, Chen Ya2li Secondary structures in solution and denaturation dynamics of flammulin Chin J Biochem Mol Biol),2000,16(4) :487 493 4 Gilliland GL,Davies D R Protein crystallization : the growth of large2 scale single crystals Meth Enzymol, 1983,104 :370 381, 5 Lowry O H, Rosebrough N J, Farr A L, Randall R J Protein

2 : 239 measurement with the Folin phenol reagent J Biol Chem, 1951, 193 : 265 275 6 Laemmli U K Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature, 1970, 227 :680 685 7 Chiari M, Righetti P G Two2dimensional polyacrylamide gel electrophoresis immobilized ph gradient in the first dimension The state of the art and controversy of vertical versus horizontal systems Electrophoresis 1992, 13 :187 191 8 Wheat T E Principles and practice of peptide analysis with capillary zone electrophoresis Mol Biotechnol, 1996,5 :263 273 9 :, 1985 :325 326 ( Pavia D L, Lampman G M, Kriz G S Jr ed Introduction to Organic Laboratory Techniques W B Saunders Company,1976) The Fourth Editorial Board of Chinese Journal of Biochemistry and Molecular Biology ( Emeritus Editor2in2Chief) ZHANG Chang2ying(Chang2ying CHANG) ( Emeritus Members of the Board) CAI Liang2wan DU Guo2guang HUANG Cui2fen LI Yu2rui XIA Shou2xuan ZHANG Chang2ying ZHANG Shu2zheng ZHENGJi ZOU Cheng2lu (Chen2lu TSOU) ( Specially Invited Members of the Board) Dr Ray WU, Section of Biochemistry,Molecular and Cell Biology, Cornell University,NY 14853, USA Dr Robert K YU,Medical College of Georgia, Institute of Molecular Medicine and Genetics,1120 15 th Street,CB22803,Augusta,Georgia 30912,USA ( Editor2in2Chief) ZHANG Nai2heng (Associate Editors2in2Chief, 3, 3 Executive Associate Editor2in2Chief) J IA Hong2ti LIANG Kang LIU De2fu SUN Zhi2xian WANGLin2fang YANG Fu2yu YAO Ren2jie (Members of the Board, 3, 3 Executive Member of the Board,alphabetically) AO Shi2zhou CHANG Zeng2yi CHEN Hui2li CHEN Ke2ming CHEN Shi2shu CHEN Su2min CUI Zhao2chun DAI Chang2bai DONG Zhi2wei FENG Zong2chen HUANG Da2nian HUANG Pei2tang HUANG You2guo J IA Hong2ti J IN You2xin JU Tian2lin LI Jian2min LI Zai2ping LIANG Ai2hua LIANG Kang LIANG Song2ping LIANG Xi2nan LIN Qi2shui LIN Xi2feng LIU Bing2wen LIU De2fu LIU De2pei MA Jian2quan MIAO Shi2ying QIANG Bo2qin QIAO De2jun QIN Jun2chuan REN Xi2lin RU Bing2gen RUAN Kang2cheng SUN Chong2rong SUN Zhi2xian TANGJian2guo TONG Tan2jun WANG Da2cheng WANGJia2xi WANGLin2fang WANG Yu WANG Zhi2zhen WEI Qun WEN Jin2kun WU Xian2rong XU Gen2jun YAN Si2xu YANG Fu2yu YANG Kai2yu YANG Ke2gong YAO Ren2jie YE Xin2sheng YIN Zong2zhu ZHANGJian2ye ZHANG Nai2heng ZHANG Ting2fang ZENG Zhong2kui ZHOU Ai2ru ZHOU Hai2meng ZHOU Jun2mei ZHU Sheng2geng