A Ne w Method of Separation and Bioactivity Assay of Flammulin

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1 ISSN CN ΠQ Chinese Journal of Biochemistry and Molecular Biology 19 (2) : ,,,,, (, ) 3, :, DEAE2Sephadex A250, CM2Sephadex C250,, , pi 819, max 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 , 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 , pi 819, max = 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 : , : (No ) 3 Tel : (0531) ,E2mail sdu edu cn,,1972 2, Received :August 8,2002 ;Accepted :October 14,2002 Supported by Ministry of Education of China for Doctor Foundation ( No ) 3 Corresponding author Tel : (0531) E2mail sdu edu cn

2 2 : 235,, 66 %, : nm, 50 % : 1 mg, (VΠV), - 20, 6 molπl HCl h, 75 % (VΠV), - 20, (OPA), LC24A 0102 molπl Na 2 HPO 4 2NaH 2 PO 4 (ph616) ;, DEAE2Sephadex A250, : 5 molπl NaOH, h,,,, molπl NaAc2HAc (ph 516) 116, CM2Sephadex C250, : 60,, NaAc2HAc 012 ml ( Πml) ( molπl), 6, 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 ( 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 ( 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

$236 19 Fig 1 Improved Ion2exchange column chromatography purification of flammulin The active fractions from DEAE2Sephadex2A250 column chromatography were applied to a$ $gradient of 0 1 molπl NaCl in 0102 molπl Na 2 HPO 4 2NaH 2 PO 4, ph 718 The activities of the fractions were determined as described under Materials and Methods Fig 3$ $Pharmacia 2 Active fractions Fig 2, Watanabe, 2 ( Fig 3 5) 23 700, pi 819 (Fig 5), Fig 5 Capillary zone electrophoretogram for flammulin Running voltage 25 kv,$

3 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) , 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

4 2 : 237 Molisch, 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 Fig 7 max 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 mgπkg 5 mgπkg 10 mgπkg ( Table 2) S 180 S mgπkg, %, 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 ( P < 0101) ( Table 5) Average survival Control 1 10Π days Ratio of the survival period prolongation( %) Value of P Π < Π < 0 01 Control 2 10Π Π < Π < 0 02

5 Table 3 Effect of flammulin on tumor S 180 in mice Group flammulin, Πmg kg miceπg solid tumor in mice Πg Control Tumor2 inhibiting ratio ( %) Value of P Table 4 Effect of flammulin on Ehrlich ascites tumor in mice Group flammulin, Πmg kg - 1 miceπg, 10 solid tumor in miceπg Control Tumor2 inhibiting ratio ( %) Value of P Control Table 5 Effect of immune apparatus in mice treated with flammulin Group flammulin thymus solid spleen Πmg kg - 1, 10 Πmg Πmg Control Control P < 0101,compared with control group Table 6 LD 50 = mgπkg, LD 50 = mgπkg, E max = , 95 % = mgπkg, 95 % = 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 unit Error : 200 mgπkg 100 mgπ kg 80 mgπkg 20 min,,,,24 h 1 2 g,, 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) : Watanabe Y, Nakanishi K, Komatsu N Flammulin, an anti2tumor substance Bull Chem Soc, Japan, 1964,37(5) : ,, ( Feng Yong2jun, Zhang Chang2kai, Chen Ya2li Secondary structures in solution and denaturation dynamics of flammulin Chin J Biochem Mol Biol),2000,16(4) : Gilliland GL,Davies D R Protein crystallization : the growth of large2 scale single crystals Meth Enzymol, 1983,104 : , 5 Lowry O H, Rosebrough N J, Farr A L, Randall R J Protein

6 2 : 239 measurement with the Folin phenol reagent J Biol Chem, 1951, 193 : Laemmli U K Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature, 1970, 227 : 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 : Wheat T E Principles and practice of peptide analysis with capillary zone electrophoresis Mol Biotechnol, 1996,5 : :, 1985 : ( 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, 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

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