S ep a r a t i on of P r ot ei ns i n A queous Tw o2p h as e S ys t e ms wi t h Hi g h2sp eed Cou nt e r2cu r re nt Ch r o m a t og r ap h y

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1 J anuary 2005 Chinese J ournal of Chromatography Vol. 23 No ( ; ) : 1000 ( PEG1000) % PEG % 016 ml/ min 900 r/ min 3313 % p H 912 PEG10002 C p H 1410 %PEG % 110 ml/ min 850 r/ min 3 p H %PEG % 110 ml/ min 850 r/ min 100 %100 % 60 % 90 % : ; ; :O658 :A : (2005) S ep a r a t i on of P r ot ei ns i n A queous Tw o2p h as e S ys t e ms wi t h Hi g h2sp eed Cou nt e r2cu r re nt Ch r o m a t og r ap h y ZHI Wenbo 1 D ENG Qiuyun 2 SONG J iangnan 1 GU Ming 1 OU YANG Fan 1 ( 1. National Key Labor atory of Biochemical Engi neeri ng Instit ute of Process Engi neeri ng The Chi nese Aca de my of Sciences Beiji ng Chi na ; 2. TAU TO Biotech Co. L TD S ha nghai Chi na) A bs t r act : High2speed counter2current chromatograp hy ( HSCCC) is a continuous li quid2liquid par2 tition chromatograp hy wit h remarkable advanta ges of high separation efficiency and no adsorption or denaturation by solid p hase. The retention of stationar y p hase and t he separation of p roteins in polyet hylene glycol 1000 ( PEG1000)2p hosp hate aqueous two2p hase s ystem (A TPs ) were studied wit h a multi2column high speed2counte r2cur re nt chromatograp h. The flow direction and speed of t he mobile p hase and t he rotation direction and s peed of t he apparatus showed different effects on t he retention of t he stationar y p hase which reached t he maximum at 3313 % wit h a flow rate of 016 ml/ min and a rotation speed of 900 r/ min in 1410 %PEG %p hosp hate A TPs. Distinct differences in partition coefficients amon g cytochrome C lysozyme and hemoglobin were found at p H 912 and t hese t hree p roteins were successf ull y separated in 1410 % PEG % p hosp hate A TPs at p H 912 by HSCCC wit h t he apparatus rotating at 850 r/ min and t he mobile p hase flow rate of 110 ml/ min. The major p rotein components in hen egg white including ovalt ransferrin oval2 bumin and l ysozyme also s how distinct diff e re nces of partition coefficie nts in P EG10002p hosp hate A TPs at p H 912. Ovalbumin and l ysozyme were successf ull y p urified to homogeneit y and ovalt rans2 ferrin to ca 60 % p urit y f rom t he hen e gg white sample wit h yields over 90 % in 1510 % PEG % p hosp hate A TPs at p H 912 wit h t he apparatus rotating at 850 r/ min and mobile p hase flow rate of 110 ml/ min. Key w or ds : high2speed counter2current chromatograp hy ; aqueous two2p hase s ystem ; p rotein ; separation and p urification : : E2mail com. : Tel : (010) Fax : (010) E2mail ipe. ac. cn.

2 1 : 13 ( high2speed counte r2cur re nt chromatograp hy HSCCC) 2 C [ 1 2 ] ; ; [ 11 ; ] HSCCC [ 3 ] 1 HSCCC [ 4 / ] 1. 1 TB E2300V ( ) ( 216 mm) ( 70 % %) ( 1 ) 120 ml [ 5 6 ] ( HX21050 [ 7 ] ) 20 ml [ 4 ] HSCCC r/ min ΦKTA PRIM E (Amersham Bio2 science Sweden) HSCCC [ 8 ] [ 9 ] [ 4 ] HSCCC TB E2300V Amer2 sham A KTA PRIM E / 1 TB E2300V Fi g. 1 Sche matic of hi gh2speed cou nter2cur rent chromat ograp hic model TB E2300V a. ΦKTA TM prime ; b. TB E2300V ; c. water bath ; 1 2 and 3. separation columns. HSCCC ( PEG1000) ( ) : ( ) [ 2 ] : C ( ) ( [ 4 ] ) ( ) ( ) ( ) Sigma (USA) [ 10 ] ( M r ) : M r ( ) B ( M r = ) (BSA M r = ) ( M r = )

3 14 23 ( M r = ) ( M r = ) V t R = ( V t ( M r = ) 1. 3 Bradford [ 12 ] PEG nm mg 2 ml 1. 9 [ 13 ] ml 280 nm A ( (SDS2PAGE) ) 100 g/ L K ( K BandScan (V4. 30 Glyko) K = A U / A L U L ) mg C 5 mg 20 mg ml 2 PEG Awad [ 11 ] : 2 [ 14 ] 014 mol/ L NaCl 10 mmol/ L 2 PEG mol/ L Tris2HCl (p H 910) PEG1000 [ 15 4 ml ] PEG ( ) HSCCC ( ) ; ; ; HSCCC 1 4 ; V e R No. rotation 1 Ta ble 1 Eff ects of f l ow di recti on of mobile p hase a nd r ot ati on di recti on of t he Status flow app a r at us on t he retenti on of s t ati ona r y p hase Rotation speed/ ( r/ min) - V e ) / ( V t ) Flow rate/ ( ml/ min) Retention/ % Flow2away/ ( ml/ h) Baseline 1 forward backward > 5 unstable 2 backward backward > 5 unstable 3 forward forward < 0. 5 stable 4 backward forward < 0. 5 stable

4 1 : ( a) ( b) Fi g. 2 Eff ects of r ot ati on speed of t he app a r at us ( a) a nd f l ow r ate of mobile p hase ( b) on t he retenti on of s t ati ona r y p hase i n 1410 % P EG % p hosp hate A TPs a. flow rate : 016 ml/ min ; flow direction : forward. b. rotation speed : 900 r/ min rotation direction : backward. 22a (600 p H p H 750 r/ min) 20 % 850 r/ min 30 % PEG10002 p H ml/ min 33 % 25 % HSCCC (30 % 50 %) / (40 % 85 %) [ 4 ] 3 p H P EG10002 Fi g. 3 Eff ect of p H on t he p r otei n p a r titi on coeff icients i n P EG10002p hosp hate s ys te m 1. cytochrome C; 2. hemoglobin ; 3. lysozyme. 3 C 3 p H C p H [ 16 ] p H S p H p H HS CCC p H p H p H p H

5 16 23 p H 912 PEG10002 C ( C ) 5 P EG10002 p H Fi g. 5 Eff ect of p H on t he p r otei n i n a hen egg w hite s a mple p a r titi on coeff icients i n P EG p hosp hate s ys te m 1. ovaltransferrin ; 2. ovalbumin ; 3. lysozyme. 4 3 Fi g. 4 HS CCC chromat ogr a m of a mi xt u re of cyt ochrome C he mogl obi n a nd l ys ozyme Conditions : separation column 120 ml coiled PTFE tube ( i1d1 216 mm ) ; mobile phase lower phase ; flow rate 110 ml/ min ; rotation speed 850 r/ min ; sample solution 2 ml con2 taining 1 mg cytochrome C 5 mg hemoglobin and 20 mg lysozyme ; solvent system : p H % ( w/ w) polyethylene glycol % (w/ w) dipotassium hydrogen phosphate in dis2 tilled water. 1. cytochrome C; 2. lysozyme ; 3. hemoglobin ; SR : stationary phase backward elution. 4 3 C ( R s ) 1115 ( ) ( 4 SR) 2. 3 p H HS CCC 4 [ 11 ] : 3 ( ) p H PEG p H 912 PEG10002 p H 912 PEG ( ) ( 7) ; ( ) 6 Fi g. 6 HS CCC chromat ogr a m of a hen egg w hite s a mple Conditions : sample solution 4 ml hen egg white prepara2 tion ; solvent system : p H % ( w/ w) polyethylene glycol % ( w/ w) dipotassium hydrogen phosphate in distilled water. Other conditions as in Fig ovaltransferrin ; 2. ovalbumin ; 3. lysozyme PEG10002 ( 7 4) ( ( > 10) p H 7 5) [ 17 ] p H 912 ( 7 3) M r

6 1 : r/ min 3313 % [ 11 ] HSCCC p H 60 % Awad p H 912 C [ 11 ] p H %PEG % ( 7 6) 110 ml/ min 850 r/ min ovalstatin C p H 912 [ 11 ] 1510 % PEG % BandScan ml/ min 850 r/ min 100 % 17 % % 90 % 1 % 550 mg ( 6 ml) 90 mg ( 95 %) 283 mg ( 94 %) 51 mg ( 93 %) [ 10 ] Tan Tianwei Shen Zhongyao. Microbiology ( 7 SDS2PA GE. Fi g. 7 ) (6) : 368 SDS2PA GE res ults of el uti on pea ks of hen egg w hite by HS CCC [ 11 ] Awad A C Moreau S Moll D Brul G Maubois J L. J Lane 1 : protein marker ; lane 2 : hen egg white sample ; lane Chromatogr A (2) : : peak 1 in Fig. 6 ; lane 4 : peak 2 in Fig. 6 ; lane 5 : peak 3 in [ 12 ] Bradford M M. Anal Biochem (2) : 248 Fig. 6 ; lane 6 : stationary phase. [ 13 ] Sambrook J Fritsch E F Maniatis T. Molecular Cloning. 2nd 3 : % PEG % [ 1 ] Ito Y Bowman R L. J Chromatogr (2) : 189 [ 2 ] Ito Y. Countercurrent Chromatography : Theory and Practice. New York : Marcel Dekker [ 3 ] Dai Deshun Wang Yiming Luo Guoan. Chinese J ournal of Analytical Chemist ry (. ) (5) : 586 [ 4 ] Zhang Tianyou. The Technology of Countercurrent Chromato2 graphy. Beijing : Science and Technology Press (.. : ) [ 5 ] Diamond A D Hsu J T. Adv Biochem Eng/ Biotechnol (1) : 89 [ 6 ] Ohlsson R Hentschel C C Williams J G. Nucleic Acids Res (2) : 583 [ 7 ] Alberttson P A. Partition of Cell Particles and Macromolecules. 3rd ed. New York : Wiley [ 8 ] Lee Y W. In : Menet J M Thibaut D eds. Countercurrent chromatography. New York : Marcel Dekker [ 9 ] Shinomiya K Kabasawa Y Yanagidaira K Sasaki H Muto M Okada T Ito Y. J Chromatogr A (122) : 103 ed. New York : Cold Spring Harbor Laboratory Press ml/ min HSCCC [ 17 ] lat Technol (13) : [ 14 ] Shibusawa Y Ito Y. J Liq Chromatogr ( 15216) : [ 15 ] Shibusawa Y Yamaguchi M Ito Y. J Liq Chromatogr Relat Technol (122) : 121 [ 16 ] Ito Y Yang F Q Fitze P Powell J Ide D. J Chromatogr A (122) : 71 Shibusawa Y Lino S Shindo H Ito Y. J Liq Chromatogr Re2