ISSN 100727626 CN 1123870ΠQ 2005 2 Chinese Journal of Biochemistry and Molecular Biology 21 (1) :101107 ( Dityophora duplicata) 1) 3 2) ( 1) 350007 ; 2) 350001) DEAE2Sepharose Sephadex G2100 ( Dityophora duplicata ( Bosc) Fischer lectin) DDFL DDFL PAGE SDS2PAGE 2213 kd Sephadex G2100 4513 kd DDFL IEF 3192 4 S 180 2 2 N2 DDFL 17 N DDFL 60 10 min ph 410910 Mg 2 + Ca 2 + Mn 2 + Zn 2 + DDFL 70163 mgπkg Q5 Purification and Characterization of Lectin from Dityophora duplicata LIN Yu2Man 1) 3 SU Ai2Hua 2) ( 1) College of Chemistry and Materials Science Fujian Normal University Fuzhou 350007 China ; 2) Fujian Health School Fuzhou 350001 China) Abstract The lectin DDFL from the fruiting bodies of Dityophora duplicata ( Bosc) Fischer was isolated and purified by physiological saline extraction (NH 4 ) 2 SO 4 precipitation and DEAE2Sepharose Sephadex G2100 chromatography purification It turned out to be a single band on PAGE The relative molecular mass of DDFL was 45 3 kd tested by gel filtration and the subunit was 2213 kd determined by SDS2PAGE its pi was about 3192 by isoelectrio focusing This lectin could agglutinate erythrocytes from rabbit calf duck chicken crucian frog and 4 types of human erythrocytes but did not agglutinate erythrocytes from turtle It also agglutinated the peripheral lymphocytes cells and S 180 tumor cells from mouse The hemagglutination on rabbit erythrocytes was inhibited by lactose raffinose galactose 2methyl2galactose 2methyl2galactose and N2acetyl2 galactosamine The amino acid composition analysis showed that it contained 17 kinds of amino acid in which the contents of aspartic acid serine phenylalanine and alanine were relatively high The N2terminal of DDFL was glycine DDFL had certain thermal2stable acid2stable and alkali2stable it still had high activity after being heated 10 minutes in 60 and had high activity within ph 410910 Its activity was depended on Mg 2 + and Ca 2 + not on Mn 2 + and Zn 2 + DDFL was toxic to mouse by celiac injection with a LD 50 of 70163 mgπkg :2004202217 :2004205217 (NoJB00029) (No CJ2057) 3 Tel : (0591) 83465225 ; E2mail : ymlin @fjnu edu cn Received :February 17 2004 ; Accepted : May 17 2004 Supported by the Education Department of Fijian Province (NoJB00029) and Testing Foundation of Fijian Colleges and Universities(No CJ2057) 3 Corresponding author Tel : (0591) 83465225 E2mail : ymlin @fjnu edu cn
102 21 Key words lectin Dityophora duplicata ( Bosc) Fischer isolation and purification characterization Pharmacia ;Ampholine ph 31510 [1 ] 1888 Stillmark H LKB ; N2 100 Sigma 112 DDFL (400 g) 60 % 4 (1500 g) PBS (0105 molπ [24 ] L - 0115 molπl NaCl ph 712) SO 2-4 DEAE2Sepharose (116 cm 30 cm) PBS PBS (0105 molπl - 015 molπl NaCl ph 712) PB (0105 molπl ph 712) [5 50 ] LKB 278 nm 60 Sephadex G2 100 (216 cm 60 cm) PBS DDFL 113 DDFL [8 ] Tris2 (ph 813) 715 % R2 [6 7 ] 250 2Schiff 114 [2 ] V 25 l PBS 25 l 1 111 PBS 25 l 2 % 2 h A B AB O [2 ] 115 11511 Sephadex G2100 S 180 : (67 kd) ; (43 kd) (25 kd) Sephadex G2100 DEAE2Sepharose (1317 kd) K av = ( V e - V 0 )Π( V t
1 : ( Dityophora duplicata) 103 - V 0 ) V 0 V t V e 2 h V e 1112 DDFL K av K av 600gΠml K av = - b lgm + C 150 l K av 30100 10 10 min DDFL K av V DDFL (20 ) 11512 SDS2PAGE [9 ] 1113 DDFL 715 % R2250 ph 3101310 ph 310 b (94 kd) 0105 molπl 2 ;ph 410510 (67 kd) (43 kd) (30 kd) 012 molπl 2 ;ph 610810 (2011 kd) 2 (1414 012 molπl ;ph 9101010 0105 kd) Weber [10 ] molπl 2 ;ph 1110 01025 molπl 2 ;ph 12101310 116 [11 Folin2 ] 117 [12 25 Ashwell 2 ] l ( ph ) 25 l 400 mgπl 014 016 018 110 112 114 116 118 ml H 2 O 210 ml 6 % 110 ml H 2 SO 4 510 ml 10 min 20 min A 490 EDTA2Na 2 A 490 V 10 mmolπl CaCl 2 A 490 MnCl 2 ZnCl 2 MgCl 2 25 l 118 EDTA2Na 2 DDFL 6 molπl HCl 110 24 h 25 l 25 l 2 h HITACH 835250 [13 ] DDFL EDTA2Na 2 119 N Chang [14 ] 1110 DDFL 50 20 g LKB 5 10 ; DDFL Ampholine ph 31510 418 % ( LD 0 ) ( LD 100 ) ph 1111 0105 molπl 2 DDFL 600g/ m1 V DDFL 25 l 2 % 2 h 1114 DDFL DDFL 20 mmolπl EDTA2Na 2 1115 [15 ] r r = lg - 1 [ (lgld 100 - lgld 0 )Π( n - 1) ] n [ 2 ] 0 2 DDFL 3 d molπl V DDFL ( 50 % ) 2 %
104 21 1116 Alsever 3 2 % 1117 1 10 7 1118 2 1 10 7 S 180 2 211 DDFL (400 g) 292 mg 01073 % DEAE2Sepharose PBS PBS 500 ml ( ) PB 500 ml ( ) 3 (Fig11) 1 A 278 ; Sephadex G2100 2 ( Fig12) 1 2 1 DDFL 38 mg 13 % 212 (DDFL) PAGE ( Fig13 A ) DEAE2 Sepharose Sephadex G2100 PAGE ( Fig13 B) 2Schiff 213 DDFL 21311 DDFL Sephadex G2100 DDFL 4513 kd ; 1 % SDS2PAGE DDFL DDFL Fig 1 DEAE2Sepharose chromatography of precipitate from 60 % saturation of ( NH 4 ) 2 SO 4 eluted with NaCl gradient ( 0 015 molπl) in PB A 278 ; Titer of agglutination initial concentration 600 gπml Fig 2 Sephadex G2100 chromatography of DDFL eluted with PBS Titer of agglutination initial concentration 600 gπml Fig 3 PAGE of DDFL Staining with Coomassie brilliant blue R2250 A : Crude lectin ; B : Purified lectin
1 : ( Dityophora duplicata) 105 DDFL Table 2 Inhibition of hemagglutinating of DDFL by sugars 2213 kd(fig14) DDFL Fig 4 SDS2PAGE of purified DDFL 11DDFL ;21Protein molecular mass marker 21312 N DDFL ( Table 1) 17 70 Chang N 90 DDFL 21313 2 DDFL Table 3 Effect of temperature on hemagglutinating activity of Table 1 Amino acid composition of DDFL DDFL Amino acids Amino acid content (g per 100 g protein) Amino acids Amino acid content (g per 100 g protein) Asp 13146 Thr 6153 Ser 9100 Glu 6123 Gly 4195 Ala 7183 Cys 3123 Val 5167 Ile 5104 Leu 3174 Tyr 3187 Phe 9123 Lys 6123 Arg 1149 Pro 5176 His 0100 Met 3123 Trp 1162 21314 DDFL pi 3192 ; 21317 DDFL DDFL 21315 DDFL 600gΠml ph 3101310 Table 2 Table 2 DDFL Table 4 Table 4 ph N2 410910 ; ph 310 2 2 1010 1110 ;ph 11 9 DDFL DDFL DDFL 21316 DDFL DDFL 21318 DDFL 600gΠml DDFL (Table 5) ( Table 3) DDFL 30 60 Sugars Inhibition Minimum inhibition concentration/ mmol Gal + 5010 Man - Fuc - Ara - Fru - Xyl - GlcNAc - GalNAc + 1215 Rha - Lac + 6125 Sur - Glc - Raf + 50 2MeGal + 25 2MeGal + 25 L - 1 Concentration of DDFLΠg ml - 1 TemperatureΠ 300 150 75 3715 18175 9138 4169 2135 1118 0159 0130 Room temper2 ature (20) + + + + + + + + + + - 30 + + + + + + + + + + - 40 + + + + + + + + + + - 50 + + + + + + + + + + - 60 + + + + + + + + + + - 70 + + + + + + + - - - - 80 + + + + - - - - - - - 90 - - - - - - - - - - - 100 - - - - - - - - - - - + :hemagglutinating - :non2hemagglutinating The same as the below table :DDFL
106 21 DDFL EDTA2Na 2 ; Table 6 Agglutinating activity of DDFL DDFL Mg 2 + Ca 2 + Zn 2 + Mn 2 + Ca 2 + Mg 2 + 0131310 mmolπl DDFL Mn 2 + Zn 2 + DDFL Ca 2 + Mg 2 + Table 4 Effect of ph on hemagglutinating activity of DDFL ph Concentration of DDFLΠ g ml - 1 300 150 75 3715 18175 9138 4169 2135 1118 0159 0130 310 + + + + - - - - - - - 410 + + + + + + + + + - - 510 + + + + + + + + + + - 610 + + + + + + + + + + - 710 + + + + + + + + + + - 810 + + + + + + + + + + - 910 + + + + + + + + - - - 1010 + + + + + - - - - - - 1110 + + + - - - - - - - - 1210 - - - - - - - - - - - 1310 - - - - - - - - - - - Table 5 Effect of metal ions on hemagglutinating activity of DDFL Lectin without treatment with EDTA2Na 2 (contrast) Lectin treated with EDTA2 Na 2 (without metal ion) Concentration of metal ionsπmmol L - 1 10 5 215 1125 01625 01313 01156 01078 + + + + + + + - - - - - - - - - Mg 2 + + + + + + + - - Ca 2 + + + + + + + - - Zn 2 + - - - - - - - - Mn 2 + - - - - - - - - 214 DDFL 21411 DDFL DDFL 600gΠml ( Table 6) Table 6 Erythrocyte Titer of agglutination Human A type 2 6 B type 2 6 AB type 2 6 O type 2 7 Rabbit 2 10 Calf 2 10 Duck 2 12 Chicken 2 8 Crucian 2 5 Turtle - Frog 2 4 Table 7 The agglutination of DDFL to lymphocyte and tumor cell of mice Titer of Minimum agglutination Cells agglutination concentrationπ g ml - 1 Spleen lymphocyte 2 6 9138 S 180 sarcoma cell 2 9 1118 21413 Table 8 LD 0 = 35 mgπkg LD 100 = 180 mgπ kg r = 11506 lgld 50 = 11849 lg - 1 11849 = 70163 mgπkg LD 50 = 70163 mgπkg 95 % = 5514189195 mgπkg Table 8 Toxic activity of DDFL to mouse Dose Number Number Death Probability lg(dose) Πmg kg - 1 of mice of death ratio ( %) unit 35 11544 10 0 0-53 11724 10 2 20 4116 80 11903 10 7 70 5152 120 21079 10 9 90 6128 180 21255 10 10 100-3 DDFL DDFL 2Schiff 0115gΠ 2 ml 3715 DDFL DDFL gπml DDFL 17 21412 DDFL DDFL 600gΠml V Table 7 Table 7 DDFL S 180
1 : ( Dityophora duplicata) 107 DDFL 15 2 2 N2 DDFL DDFL DDFL DDFL [2 16 ] DDFL DDFL S 180 ; ; duplicata DDFL Lin [17] VAG LD 50 17 5 mgπkg [4] AAVP LD 50 15 85 mgπkg DDFL VAG AAVP LD 50 70163 mgπkg Mg 2 + Ca 2 + Zn 2 + Mn 2 + DDFL Ca 2 + Mg 2 + Mn 2 + Zn 2 + ( References) 1 Peumans W J Van Damme J E M1Lectin as plant defense protein Plant Physiol 1995 109 :347352 2 : (Su Ce Zhu Su2Fang Wu Yao2Sheng Deng Yong Zhou De2Yi Purification and characterization of lectin from the seeds of Artocarpus lingnanensis Chin J Biochem Mol Biol) 1999 15 (1) :142144 4 ( Agrocybe aegerita) ( Sun Hui Zhao Chen2Guang Tong Xin Qi Yi2Peng Purification and characterization of a novel lectin AAVP fromfruiting bodies of the edible fungus Agrocybe aegerita Chin J Biochem Mol Biol) 2003 19(1) :96 Zheng Mo Han2Qing ed Lectin Beijing : Science Press) 1986 :1 agglutinins from martine algae Comp Biochem Physiol 1989 94b :107 136 111 3 17 Lin J Y Chou T B Isolation and characterization of a lectin from edible ( Zhang Cheng2Lu Zhou mushroom Volvariella volvacea J Biochem 1984 96 (1) :3540 102 5 Wang H X Bun T N Ooi V E C Lectins from mushrooms Mycol Res 1998 102(8) :897906 6 Dd2S3P (Lin Yu2Man Isolation Purification and characterization of polysaccharide Dd2S3P from Dictyophora duplicata ( Bosc ) Chin Biochem J) 1997 13 (1) :99102 Fischer 7 DdM2S ( Lin Yu2Man Isolation purification and characterization of polysaccharide DdM2S from the submerged mycelium of Edible Fungi Chin) 2003 22(6) :5253 Dictyophora 8 : ( Wu He2Ling Lin Jin2Hu ed Experimental Techniques in Genetics Beijing : Higher Education Press) 1984 : 260263 9 Laemmli U K Cleavage of structural proteins during the assembly of the head of bacteria phage T4 Nature 1970 227(5259) : 680685 10 Weber K Osborn M The reliability of molecular weight determinations by dodecyl sulfate polyacrylamide gel electrophoresis J Biol Chem 1969 244(16) : 44064412 11 : (Chen Yue2Quan ed Experimental Methods and Techniques in Biochemistry Beijing : Science Press) 2002 :164166 12 Ashwell G New colorimetric methods of sugar analysis VII The phenol2 sulfuric acid reaction of carbohydrates Methods Enzymol 1966 8 : 93 96 13 Edelhoch H Spectroscopic determination of tryptophan and tyrosine in proteins Biochemistry 1967 6 :19481954 14 Chang J Y Micro2sequence analysis of peptides and proteins using 42N N2dimethylamino benzene 4 2isothio2cyanateΠphenylisothiocyanate double coupling method FEBS Lett 1978 93 (2) :205211 15 : ( Xia Bing2Nan Sun Rui2Yuan Zhang Jia2Quan ed Handbook of Pharmaceutical Experiment 1986 :5675 Guiyang : Guizhou People s Press) 16 Chiles T C Bird K T A comparative study of animal erythrocyte