- Email: [email protected]
Chapter 21 Peptides and Proteins
365
Chapter 2 1 PEPTIDES AND PROTEINS P r o t e i n s , t h e e s s e n t i a l components o f l i v i n g m a t t e r , a r e composed o f p o l y p e p t i d e c h a i n s . Enzymes and a number o f hormones a r e s t r u c t u r a l l y based on p r o t e i n s . A knowledge o f t h e amino a c i d sequence i n p e p t i d e s and i n p r o t e i n s i s v e r y i m p o r t a n t i n b i o c h e m i c a l r e s e a r c h . The number o f p u b l i s h e d a p p l i c a t i o n s o f l i q u i d chromatography t o t h e s e p a r a t i o n o f p e p t i d e s and p r o t e i n s i s enormous and perhaps outnumbers t h e sum o f l i q u i d chromatography a p p l i c a t i o n s t o t h e s e p a r a t i o n o f a l l t h e o t h e r compounds. I t would be i m p o s s i b l e h e r e t o g i v e a r e v i e w o f a l l s e p a r a t i o n methods i n which g r a d i e n t e l u t i o n has been a p p l i e d t o t h e chromatography o f p e p t i d e s and p r o t e i n s . T h e r e f o r e , a b r i e f survey o f some g e n e r a l l y a p p l i c a b l e methods employing t h e g r a d i e n t e l u t i o n t e c h n i q u e i s g i v e n and i l l u s t r a t e d , b y o n l y a few examples, t o g i v e a p i c t u r e o f what g r a d i e n t e l u t i o n l i q u i d column chromatography means i n s t u d i e s o f t h e s e compounds. A s y s t e m a t i c , comprehensive survey o f l i q u i d chromatography a p p l i c a t i o n s i n t h i s f i e l d may be f o u n d elsewhere (see, f o r example, r e f s . 1 - 3 ) . A p p l i c a t i o n s o f l i q u i d chromatography t o t h e p r e p a r a t i v e p u r i f i c a t i o n and f r a c t i o n a t i o n o f p r o t e i n s a r e i m p o r t a n t . High-performance l i q u i d chromatography (HPLC) has b r o u g h t new dimensions t o t h e a n a l y s i s o f p r o t e i n s , p e p t i d e s and amino a c i d s w i t h r e s p e c t t o speed o f a n a l y s i s , s e p a r a t i o n e f f i c i e n c y and s e n s i t i v i t y , which makes i t p o s s i b l e t o a c h i e v e m i c r o - s c a l e s e p a r a t i o n s o f complex m i x t u r e s of p e p t i d e s i n s m a l l amounts o f samples i n l e s s t h a n 1 h, i n c o n t r a s t 133 t o e a r l i e r e l a b o r a t e and time-consuming methods g i v i n g l o w y i e l d s o f m a t e r i a l
.
To d e t e r m i n e t h e amino a c i d c o m p o s i t i o n o f p r o t e i n s and p e p t i d e s , i t i s necessary f i r s t t o s u b j e c t t h e s e compounds t o t o t a l o r s e q u e n t i a l h y d r o l y s i s . I n t o t a l h y d r o l y s a t e s t h e r a t i o o f t h e i n d i v i d u a l amino a c i d s can be determined, b u t s e q u e n t i a l h y d r o l y s i s a l l o w s t h e d e t e r m i n a t i o n o f t h e sequence o f amino a c i d s i n p e p t i d e c h a i n s . Edman d e g r a d a t i o n i s a n o n - d e s t r u c t i v e ,
subtractive
method o f p r o t e i n a n a l y s i s u s i n g r e p e a t e d d e g r a d a t i o n o f p e p t i d e s o r p r o t e i n s i n three-step cycles. I n the f i r s t step, t h e peptide o r p r o t e i n reacts w i t h phenyl i s o t h i o c y a n a t e t o g i v e t h e phenyl t h i o c a r b a m y l (PTC) d e r i v a t i v e . I n t h e second step, t h e d e r i v a t i z e d N - t e r m i n a l amino ac d i s h y d r o l y s e d f r o m t h e remainder o f t h e p r o t e i n o r p e p t i d e and i t s 2-an l i n o - 5 - t h i a z o l i n e d e r i v a t i v e i s formed and c o n v e r t e d i n t o t h e s t a b l e 3-phenyl 2 - t h i o h y d a n t o i n (PTH) d e r i v a t i v e References on p . 376.
366 i n t h e t h i r d step. Because o n l y t h e N-terminal amino a c i d i s cleaved i n t h i s procedure and t h e remainder o f t h e p e p t i d e o r p r o t e i n remains i n t a c t , t h e whole procedure can be repeated as many times as necessary t o f i n d t h e amino a c i d sequence. L i q u i d chromatography i s v e r y s u i t a b l e f o r combination w i t h Edman degradation as a r a p i d and r e l i a b l e a n a l y t i c a l technique t h a t makes i t p o s s i b l e t o p u r i f y and t o check t h e p u r i t y of o r i g i n a l p r o t e i n s o r p e p t i d e s and o f PTC d e r i v a t i v e s formed i n t h e f i r s t s t e p of Edman degradation, t o separate, i d e n t i f y and quant i t a t e PTH-amino a c i d d e r i v a t i v e s formed i n each t h r e e - s t e p Edman degradation cycle. I n a l l o f these l i q u i d chromatography a p p l i c a t i o n s g r a d i e n t e l u t i o n , e i t h e r continuous o r stepwise, may be used. I n t h e subsequent d e t e r m i n a t i o n o f t h e amino a c i d sequence i n p r o t e i n and p e p t i d e hydrolysates, t h e methods f o r amino a c i d a n a l y s i s a r e a p p l i e d ; t h e a p p l i c a t i o n s o f t h e g r a d i e n t e l u t i o n technique f o r t h i s purpose were t r e a t e d i n Chapter 20. P r i o r t o t h e d e t e r m i n a t i o n o f t h e amino a c i d r a t i o and/or sequence o r f o r o t h e r biochemical research, i t i s necessary t o p u r i f y and separate p r o t e i n s and peptides i n mixtures, and l i q u i d column chromatography i s u n i q u e l y s u i t e d f o r t h i s purpose. However, t h e s p e c i f i c f r a g i l e c h a r a c t e r o f t h e biopolymers should be borne i n mind when s e l e c t i n g t h e chromatographic c o n d i t i o n s , i n o r d e r t o avoid the r i s k o f possible denaturation o f proteins. This r i s k i s p a r t i c u l a r l y important w i t h enzymes, where t h e i r s p e c i f i c topography, which g i v e s r i s e t o t h e i r p a r t i c u l a r a c t i v i t i e s , may become l o s t by s t r u c t u r a l changes when t h e
.
enzyme i s brought i n t o c o n t a c t w i t h an u n s u i t a b l e column packing m a t e r i a l 3 I n numerous e a r l i e r a p p l i c a t i o n s o f l i q u i d chromatography t o t h e p u r i f i c a t i o n and f r a c t i o n a t i o n o f peptides and p r o t e i n s , f r a c t i o n s o f t h e e l u a t e were c o l l e c t e d and t h e amount o f compounds e l u t e d was determined u s i n g spectrophotom e t r i c methods, measurement o f t h e r a d i o a c t i v i t y o f l a b e l l e d compounds, measurement o f enzymatic a c t i v i t y , e t c . I n more r e c e n t a p p l i c a t i o n s , m a i n l y those u s i n g e f f i c i e n t columns, t h e e l u a t e s a r e monitored c o n t i n u o u s l y . The d e t e c t i o n o f aromatic peptides employs U V a b s o r p t i o n a t 280 nm. U V detect i o n a t 254 nm has o f t e n been u t i l i z e d i n t h e chromatography o f p r o t e i n s , hormones and enzymes based on p r o t e i n s . Non-aromatic peptides were detected i n t h e U V r e g i o n between 200 and 210 nm. Enzymes i n t h e e l u a t e can be detected by continuous measurement o f t h e i r enzymatic a c t i v i t y and l a b e l l e d compounds by continuous m o n i t o r i n g o f r a d i o a c t i v i t y , Post-column d e r i v a t i z a t i o n i s o f t e n used f o r t h e d e t e c t i o n o f p r o t e i n s and peptides. D e r i v a t i v e s w i t h o-phthalaldehyde o r o t h e r reagents a r e d e t e c t e d i n t h e column e f f l u e n t s employing flow-through r e a c t o r s connected w i t h f l u o r i m e t r i c d e t e c t o r s o r w i t h photometric d e t e c t o r s o p e r a t i n g i n t h e U V o r v i s i b l e region.
36 7 21.1.
ION-EXCHANGE CHROMATOGRAPHY
Ion-exchange chromatography has been most f r e q u e n t l y used i n t h e chromatography o f p e p t i d e s and p r o t e i n s . The s e p a r a t i o n o f p e p t i d e s and p r o t e i n s by ion-exchange chromatography i s based on e l e c t r o s t a t i c i n t e r a c t i o n s between t h e charged exchange groups o f t h e exchanger and t h e o p p o s i t e charge s i t e s o f t h e p e p t i d e o r p r o t e i n . The amphoteric c h a r a c t e r o f t h e s e compounds a l l o w s b o t h c a t i o n and a n i o n exchangers t o be used. A t t h e i s o e l e c t r i c p o i n t , t h e p o s i t i v e and n e g a t i v e charges o f t h e s o l u t e a r e i n e q u i l i b r i u m . A t a pH l o w e r t h a n t h a t c o r r e s p o n d i n g t o t h e i s o e l e c t r i c p o i n t , t h e p r o t e i n o r p e p t i d e i s r e t a i n e d on a c a t i o n exchanger owing t o t h e e l e c t r o s t a t i c f o r c e s , whereas i t i s u n r e t a i n e d on an a n i o n exchanger. The o p p o s i t e h o l d s t r u e a t a pH above t h a t c o r r e s p o n d i n g t o t h e i s o e l e c t r i c p o i n t . However, t h e use o f a n i o n exchangers i s o f t e n p r e f e r r e d owing t o t h e b e t t e r s t a b i l i t y o f p r o t e i n s i n t h e i r a n i o n i c forms. To improve t h e p o s s i b i l i t i e s o f p e n e t r a t i o n o f l a r g e p r o t e i n m o l e c u l e s i n t o t h e i n t e r i o r o f t h e p a r t i c l e s , exchangers w i t h wide pores a r e s e l e c t e d . Because t h e r i g i d i t y o f exchangers w i t h an o r g a n i c m a t r i x decreases and t h e i r s w e l l i n g i n c r e a s e s w i t h i n c r e a s i n g p o r e s i z e s , which i s o b v i o u s l y disadvantageous f r o m t h e p o i n t o f view o f column bed s t a b i l i t y and maximum t o l e r a b l e f l o w - r a t e s , narrow-pore i o n exchangers a r e sometimes p r e f e r r e d f o r t h e chromatography o f very l a r g e proteins. P o l y i o n s such as p e p t i d e s and p r o t e i n s a r e n o r m a l l y v e r y f i r m l y a t t a c h e d t o i o n exchangers by many i o n i c groups i n t h e i r molecules. To a c h i e v e t h e i r e l u t i o n , i t i s necessary t o employ a b u f f e r w i t h pH a b o u t 1 u n i t above ( i n c o n n e c t i o n
w i t h c a t i o n exchangers) o r below ( w i t h a n i o n exchangers) t h e i s o e l e c t r i c p o i n t o f t h e p r o t e i n o r p e p t i d e . T h e r e f o r e , optimum c o n d i t i o n s o f e l u t i o n s h o u l d be e s t a b l i s h e d f o r each compound and i t i s g e n e r a l l y necessary t o change t h e c o m p o s i t i o n o f t h e m o b i l e phase i n t h e c o u r s e o f e l u t i o n . Stepwise e l u t i o n can be used f o r t h i s purpose; however, a l l substances r e l e a s e d by a change o f b u f f e r c o m p o s i t i o n a r e e l u t e d t o g e t h e r , u n f r a c t i o n a t e d , u s u a l l y as peaks w i t h s h a r p f r o n t s and pronounced t a i l i n g . F a l s e peaks may o c c u r i f t h e b u f f e r c o m p o s i t i o n i s changed b e f o r e t h e complete e l u t i o n o f t h e t a i l i n g peak has been achieved. T h i s drawback i s a v o i d e d when u s i n g c o n t i n u o u s g r a d i e n t s f o r e l u t i o n , w h i c h y i e l d s symmetrical peaks, b e t t e r r e s o l v e d f r o m each o t h e r t h a n i n s t e p w i s e elution. During gradient e l u t i o n , t h e solutes a r e e l u t e d i n order o f t h e i r i s o e l e c t r i c p o i n t s . I n a g r a d i e n t o f i n c r e a s i n g i o n i c s t r e n g t h a t a pH c l o s e t o t h e i s o e l e c t r i c p o i n t s o f s o l u t e s , t h e s a l t i n t h e e l u e n t competes w i t h t h e s o l u t e s f o r t h e exchange groups o f t h e exchanger and t h i s competing e f f e c t i n c r e a s e s w i t h i n c r e a s i n g i o n i c s t r e n g t h o f t h e m o b i l e phase. References on p . 376.
368 The i o n i c s t r e n g t h can be changed b y changing t h e c o n c e n t r a t i o n o f t h e b u f f e r o r , more f r e q u e n t l y , by i n c r e a s i n g t h e c o n c e n t r a t i o n o f a n e u t r a l s a l t . I d e a l l y , t h e i o n i c s t r e n g t h o f t h e s t a r t i n g b u f f e r s h o u l d be low. However, b u f f e r s w i t h v e r y l o w i o n i c s t r e n g t h s have low b u f f e r i n g c a p a c i t i e s and l a r g e changes i n i o n i c s t r e n g t h may cause l a r g e changes i n t h e volumes o f s w o l l e n exchangers o f c e r t a i n t y p e s and, t h e r e f o r e , b u f f e r s w i t h v e r y l o w i o n i c s t r e n g t h s s h o u l d b e avoided. I f a pH g r a d i e n t i s a p p l i e d f o r chromatography o f p r o t e i n s o r p e p t i d e s on a cation-exchange column, t h e o r i g i n a l pH i s s e l e c t e d below t h a t c o r r e s p o n d i n g t o t h e l o w e s t i s o e l e c t r i c p o i n t o f t h e compound i n t h e sample m i x t u r e and t h e n i t i s i n c r e a s e d w i t h t i m e t o a c h i e v e t h e e l u t i o n and s e p a r a t i o n . I n c o n t r a s t ,
t h e o r i g i n a l pH above t h e i s o e l e c t r i c p o i n t s o f s o l u t e s i s s e l e c t e d i n a n i o n exchange chromatography and i t i s decreased d u r i n g t h e e l u t i o n .
resin^^-^"'
C a t i o n - o r an on-exchange polystyrene-divinylbenzene , ion exchangers based on m o d i f i e d c e l l u l ~ s e ~ -d ~e x~t r,a n (Sephadex) 24-32,130, agarose 32,33 o r g l y c o l m e t h a c r y l a t e (Spheron) g e l s 34-39 and i n o r g a n i c i o n (Sepharose) exchangers, such as h y d r o x y a p a t i t e w i t h anion-exchange
proper tie^^'-^*,
have
been w i d e l y appl ed f o r t h e p u r i f i c a t i o n and f r a c t i o n a t i o n o f p e p t i d e s and p r o t e i n s b y 1 i q u d chromatography. Most c l a s s i c a l i o n exchangers do n o t w i t h s t a n d e l e v a t e d p r e s s u r e s and t h e r e f o r e r a p i d analyses a t e l e v a t e d f l o w - r a t e s o f t h e m o b i l e phase a r e n o t p o s s i b l e . Spheron exchangers and some exchangers based on s t y r e n e - d i v i n y l b e n z e n e copolymers a r e e x c e p t i o n s f r o m t h i s r u l e . However, p r o t e i n s may become denatured by v i r t u e o f hydrophobic i n t e r a c t i o n s w i t h t h e p o l y s t y r e n e m a t r i x . R e a l l y r a p i d analyses o f p r o t e i n s and p e p t i d e s have become p o s s i b l e o n l y w i t h i o n exchangers based on m i c r o p a r t i c u l a t e i n o r g a n i c s u p p o r t s w i t h c h e m i c a l l y bonded ion-exchange f u n c t i o n a l groups, which a r e s u f f i c i e n t l y r i g i d and f o r m s t a b l e column beds a t h i g h p r e s s u r e s . An o r g a n i c s u r f a c e l a y e r bonded on t h e i n o r g a n i c s u p p o r t i s u s e f u l f o r p r o t e c t i n g p r o t e i n s f r o m d i r e c t c o n t a c t w i t h t h e i n o r g a n i c surface and c o n t r i b u t e s t o t h e chromatographic p a r t i t i o n i n g process i n a s i m i l a r manner t o ion-exchange r e s i n s o r m o d i f i e d g e l s . An i m p o r t a n t advance was t h e i n t r o d u c t i o n o f column p a c k i n g m a t e r i a l s p r e p a r e d by chemical bonding o f g l y c e r y l p r o p y l s i l a n e on t h e s u r f a c e o f c o n t r o l l e d p o r o s i t y glass. The p r o d u c t r e t a i n s i t s t e r m i n a l epoxy group, which can be s u b j e c t e d t o r e a c t i o n w i t h a n u c l e o p h i l i c reagent t o g i v e t h e required chemically modified m a t e r i a l . These m a t e r i a l s have been marketed under t h e name Glycophase/CPG. I o n exchangers o f t h i s t y p e (DEAE-, QAE-, SP- o r SM-Glycophase/CPG) 43-51 o r i o n exchangers c h e m i c a l l y bonded on a s i l i c a g e l s u p p o r t 52-55 made p o s s i b l e h i g h speed s e p a r a t i o n s o f p r o t e i n s and p e p t i d e s . Because ion-exchange chromatography can r e s o l v e p r o t e i n s w i t h s m a l l charge d i f f e r e n c e s , i t i s p o s s i b l e t o s e p a r a t e
369 isoenzymes t h a t have n e a r l y i d e n t i c a l amino a c i d sequences i n t h e s e h i g h l y s e l e c t i v e and e f f i c i e n t chromatographic systems. C o n c e n t r a t i o n ( i o n i c s t r e n g t h ) o r pH g r a d i e n t s i n m o b i l e phases a r e u s u a l l y formed f r o m s o l v e n t s c o n t a i n i n g d i f f e r e n t c o n c e n t r a t i o n s o f phosphate, a c e t a t e o r p y r i d i n e - a c e t a t e b u f f e r s i n water. J u s t t o o b t a i n an i n s i g h t i n t o t h e v a r i e t y o f a p p l i c a t i o n s p o s s i b l e , we can mention a few examples, such as chromatography o f serum p r o t e i n s on an a n i o n exchange r e s i n u s i n g a c o n c e n t r a t i o n g r a d i e n t o f a c e t a t e b u f f e r 8 ; t h e d e t e r m i n a t i o n o f reduced and o x i d i z e d g l u t a t h i o n e i n wheat f l o u r s and doughs on a c a t i o n exchange r e s i n w i t h a g r a d i e n t o f h y d r o c h l o r i c a c i d c o n c e n t r a t i o n 7 ; t h e s e p a r a t i o n o f p r o t e i n s i n human and animal s e r a by chromatography on DEAE-cellulose a n i o n exchanger u s i n g c ~ n c e n t r a t i o n lo~r pH12 g r a d i e n t s i n phosphate b u f f e r s , c o n c e n t r a t i o n gradients i n T r i s buffers16y22y23y131
o r gradients o f increasing concentration
o f sodium c h l o r i d e i n water2' ; t h e f r a c t i o n a t i o n o f t y r o s i n e - r i c h p r o t e i n s f r o m wool on a Q A E - c e l l u l o s e a n i o n exchanger u s i n g a g r a d i e n t o f i o n i c s t r e n g t h i n a l k a l i n e B-alanine buffer";
and t h e s e p a r a t i o n and p u r i f i c a t i o n o f t h e enzymes
8-glucan h y d r o l a s e 1 5 and B-N-acetyl -D-hexosaminidase19 on DEAE-cell u l o s e u s i n g an i o n i c s t r e n g t h ( c o n c e n t r a t i o n o f a l k a l i m e t a l c h l o r i d e ) g r a d i e n t i n a phosphate b u f f e r . We can f u r t h e r m e n t i o n t h e chromatography o f i n s u l i n and i t s d e r i v a t i v e s on a DEAE-Sephadex a n i o n exchanger u s i n g a c o n c e n t r a t i o n g r a d i e n t o f sodium c h l o r i d e i n a Tris-HC1 b u f f e r c o n t a i n i n g 7 m o l / l urea26; t h e chromatography o f t h y m o p o i e t i n s on QAE-Sephadex u s i n g a c o n c e n t r a t i o n g r a d i e n t of potassium c h l o r i d e 1 3 2 ; t h e f r a c t i o n a t i o n o f whey p r o t e i n s and c a s e i n i n cows' m i l k on t h e same m a t e r i a l u s i n g a c o n c e n t r a t i o n g r a d i e n t o f sodium c h l o r i d e i n a phosphate b u f f e r 2 4 ; t h e chromatography o f enzymes r i b o n u c l e a s e s on a CM-Sephadex c a t i o n exchanger u s i n g combined pH and i o n i c s t r e n g t h g r a d i e n t s i n a phosphate b u f f e r 3 1 ; t h e chromatography o f a - b u n g a r o t o x i n and o t h e r components o f snake venom on t h e same column p a c k i n g w i t h a l i n e a r c o n c e n t r a t i o n g r a d i e n t o f ammonium a c e t a t e i n w a t e r
(0.05-1 m ~ l / l ) ' ~and ~ ; t h e f r a c t i o n a t i o n o f human serum p r o t e i n s by a n i o n exchange chromatography on DEAE-Sepharose C1-6B u s i n g a l i n e a r g r a d i e n t o f i n c r e a s i n g sodium c h l o r i d e c o n c e n t r a t i o n i n 0.05 m o l / l Tris-HC1 b u f f e r a t pH 8.6
33
On Spheron i o n exchangers, s i m i l a r t y p e s o f compounds can be chromatographed as on c e l l u l o s e o r p o l y d e x t r a n exchangers, b u t Spheron m a t e r i a l s have t h e advantages o f b e t t e r mechanical r i g i d i t y and s t a b i l i t y a t h i g h e r p r e s s u r e s , which a l l o w s more r a p i d s e p a r a t i o n s t o be a c h i e v e d a t h i g h e r f l o w - r a t e s o f t h e m o b i l e phase. An example o f g r a d i e n t e l u t i o n chromatography o f human plasma on DEAE-Spheron column i s shown i n F i g . 21.1.
The e l u t i o n p r o f i l e h e r e i s s i m i l a r t o t h a t o b t a i n e d
on D E A E - c e l l ~ l o s e ~ O ~n. h y d r o x y a p a t i t e columns, f o r example, f r a c t i o n a t i o n o f myos in40y41 and
his tone^^^ was
achieved u s i n g i o n i c s t r e n g t h g r a d i e n t s i n phos-
p h a t e b u f f e r s . By chromatography on DEAE-Glycophase/CPG u s i n g 1 i n e a r c o n c e n t r a t i o n
References on p . 3 7 6 .
.
370
30
1.0
60
90
t(min) 120
[roo
70C
A,,,
100
0.6
0.2 10
40
70
F.N.
t
Y o0
%A
loo
F i g . 21.1. P r o t e i n mapping o f human plasma by ion-exchange l i q u i d chromatography on a m o d i f i e d diethylaminoethyl-Spheron anion exchanger. Column: DEAE-Spheron, 25-32 pm, 190 x 8 mm I.D. Gradient: m u l t i - l i n e a r , as i n d i c a t e d by t h e t h i n f u l l l i n e , from 0 t o 100% B i n A and then from 0 t o 100% C i n B. B u f f e r s : S o l v e n t A, 0.025 m o l / l phosphoric a c i d - T r i s (pH 8 . 5 ) ; s o l v e n t B, 0.5 m o l / l phosphoric a c i d T r i s (pH 3.5); s o l v e n t C, s o l v e n t B w i t h 1.0 m o l / l KC1. Flow-rate: 3.75 ml/min. F r a c t i o n s c o l l e c t e d a t 1.5-min i n t e r v a l s . F.N. = f r a c t i o n number; 4254 = absorbance a t 254 nm. Sample: 2 m l o f human plasma, c e n t r i f u g e d b e f o r e a p p l i c a t i o n . (Reproduced from r e f . 34 w i t h permission.) g r a d i e n t s o f sodium c h l o r i d e i n a c e t a t e o r Tris-HC1 b u f f e r s (pH 8 ) , i t was p o s s i b l e t o separate isoenzymes o f l a c t a t e dehydrogenase, hexokinase, etc.,
in
25-40 min45-50. Human serum p r o t e i n s and r a t kidney and r a t l i v e r isoenzymes c o u l d be r a p i d l y separated by anion-exchange chromatography on a P E I 6-LiChrospher S i 500-column ( a p e l l i c u l a r anion exchanger on a s i l i c a gel support) u s i n g l i n e a r
g r a d i e n t s o f sodium a c e t a t e c o n c e n t r a t i o n i n 0.02 m o l / l T r i s - a c e t a t e b u f f e r
21.2.
CHROMATOFOCUSING
pH Gradients may be generated i n an ion-exchange column i t s e l f , when i t i s o r i g i n a l l y e q u i l i b r a t e d w i t h a b u f f e r w i t h a pH d i f f e r e n t t o t h a t o f t h e b u f f e r used f o r e l u t i o n . Using a s p e c i a l b l e n d o f b u f f e r s w i t h even b u f f e r i n g c a p a c i t i e s over a wide pH range and s p e c i a l i o n exchangers, a smooth continuous pH g r a d i e n t ,,56-59
may be generated on t h e column. T h i s technique, s o - c a l l e d "chromatofocusing
i s t r e a t e d i n more d e t a i l i n P a r t 111. Very s i m i l a r i n p r i n c i p l e i s s o - c a l l e d "ampholyte-displacement
chromatography"60. An example o f t h e s e p a r a t i o n o f
s o l u b l e p r o t e i n s e x t r a c t e d from e l k muscle u s i n g t h e chromatofocusing technique on agarose-derived i o n exchangers ( P o l y b u f f e r exchangers) i s shown i n F i g . 21.2
59
.
371
0.5 A280
0.4
9 PH
0.3
8
0.2
7
0.1
0
50
100
150
200
. 6250
V(ml)
F i g . 21.2. F r a c t i o n a t i o n o f s o l u b l e p r o t e i n s e x t r a c t e d f r o m e l k muscle by c h r o matofocusing. Column: Pharmacia C 10/40; packed w i t h p o l y b u f f e r exchanger, PBE 94, bed h e i g h t = 45 mm. Column e q u i l i b r a t i o n b u f f e r , 0.025 m o l / l ethanol-amineaHC1 (pH 9.4); e l u t i o n b u f f e r , 0.0075 mmol/pH u n i t / m l P o l y b u f f e r 96 (pH 6 ) . Column s e l f - g e n e r a t e d pH g r a d i e n t i s i n d i c a t e d by t h e f u l l l i n e . F l o w - r a t e : 20 cm/h. D e t e c t i o n : UV a t 280 nm. A 0 absorbance a t 280 nm; V = e l u t i o n volume. Sample: 5 m l o f s u p e r n a t a n t f r o m ef! m i a t homogenate. (Reproduced f r o m r e f . 59 w i t h permi s s i o n . ) 21.3.
CHROMATOGRAPHY ON PDLYAMIDE GELS
Polyamide g e l s w i t h amide groups can behave as weak a n i o n exchangers and bond p e p t i d e s and p r o t e i n s by e l e c t r o s t a t i c f o r c e s . T h e r e f o r e , t h e s e compounds can be chromatographed on polyamide g e l s , such as Bio-Gel P, u s i n g e l u t i o n w i t h pH o r i o n i c s t r e n g t h g r a d i e n t s i n a c e t a t e , phosphate, Tris-HC1 o r o t h e r b u f f e r s . T h i s t e c h n i q u e was a p p l i e d , f o r example, t o s e p a r a t i o n s o f m e t h i o n y l p e p t i d e s
61
,
t h e chromatography o f i o d i n a t e d L - t y r o s y l - L - t y r o s i n e peptides6’ and t h e r e s o l u t i o n 63 o f venom p r o t e i n s
.
21.4.
CHROMATOGRAPHY ON POLAR ADSORBENTS AND ON POLAR BONDED PHASES
Chromatography on p o l a r adsorbents i s r e l a t i v e l y r a r e l y used f o r s e p a r a t i o n s o f p r o t e i n s and p e p t i d e s . UV-absorbing d e r i v a t i v e s o f p e p t i d e s ( p h e n y l t h i o h y d a n t o i n and benzoyl d e r i v a t i v e s ) were chromatographed on s i l i c a g e l u s i n g e l u t i o n w i t h a c o n t i n u o u s g r a d i e n t o f e t h a n o l i n c h l ~ r o f o r m ~p~r o, p a n o l , methanol and d i c h l o r o m e t h a n e i n n - h e ~ a n eo~r ~s t e p w i s e g r a d i e n t s o f anhydrous e t h a n o l and a c e t i c a c i d i n dichloromethane66. UV d e t e c t i o n a t 254 nm i s s u i t a b l e w i t h t h e s e systems. P e p t i d e s o f human f o e t a l g l o b i n and o f m u r i n e l a a n t i g e n s were s e p a r a t e d by chromatography on a n i t r i l e phase bonded on s i l i c a (Zorbax CN) u s i n g a c o n t i n u o u s g r a d i e n t o f o r g a n i c s o l v e n t i n aqueous a c i d i c medium (acetone i n phosphate b u f f 68 e r , pH o r propanol i n 0.1% t r i f l u o r o a c e t i c a c i d ) f o r e l u t i o n .
d7,
References on p . 376.
372 21.5.
HYDROPHOBIC INTERACTION CHROMATOGRAPHY
It has been observed t h a t i n chromatography on g e l s i n aqueous media, p r o t e i n s
and peptides a r e e l u t e d i n disagreement w i t h t h e i r expected e l u t i o n o r d e r acc o r d i n g t o t h e i r molecular s i z e s and t h a t t h e r e t e n t i o n i s i n f l u e n c e d by t h e mobile phase composition. Therefore', i t i s obvious t h a t , i n a d d i t i o n t o a p o s s i b l e s t e r i c e x c l u s i o n mechanism, o t h e r i n t e r a c t i o n s c o n t r o l t h e chromatographic process I t has been e s t a b l i s h e d t h a t these i n t e r a c t i o n s a r e hydrophobic, i.e.,
t h a t they
o r i g i n a t e i n a t t r a c t i o n f o r c e s between water molecules, by v i r t u e o f which t h e molecules of biopolymers a r e excluded from t h e m o b i l e phase on t o t h e gel surface. T h i s e f f e c t i s o f a s i m i l a r n a t u r e as i n reversed-phase chromatography on chemic a l l y bonded non-polar a l k y l o r a r y l phases. Peptides, p r o t e i n s and o t h e r b i o polymers c o u l d be separated on non-polar o r s l i g h t l y p o l a r o r g a n i c g e l s ( o r on m a t e r i a l s w i t h a c h e m i c a l l y bonded o r g a n i c l a y e r on an i n o r g a n i c support, such as G l y ~ o p h a s e - G / C P G ) ~u~s i n g t h i s s e p a r a t i o n mechanism and t h e corresponding chromatographic technique has been c a l l ed graphy" 6 9 y 7 0 .
'I
hydrophobi c i n t e r a c t i o n chromato-
As i n chromatography on c h e m i c a l l y bonded non-polar phases, r e t e n -
t i o n increases w i t h i n c r e a s i n g c o n c e n t r a t i o n o f a s a l t and decreases w i t h i n c r e a s i n g c o n c e n t r a t i o n o f an o r g a n i c s o l v e n t i n t h e mobile phase. Gels cont a i n i n g e t h e r e a l bonds and hydroxy o r e s t e r groups, such as unmodified Spheron gel72, can be used f o r t h e hydrophobic i n t e r a c t i o n chromatography o f p r o t e i n s and peptides, b u t agarose g e l s w i t h coupled hydrophobic groups, such as o c t y l , phenyl o r benzyl, have been s p e c i a l l y developed f o r t h i s purpose. The o r g a n i c s o l v e n t f o r t h e mobile phase g r a d i e n t should be s e l e c t e d w i t h c a r e and t h e s o l u b i l i t y o f p r o t e i n s i n t h e mobile phase should be borne i n mind. Most o f t e n , 73 g r a d i e n t s o f i n c r e a s i n g c o n c e n t r a t i o n o f e t h y l e n e g l y c o l (0-50%) i n a c e t a t e , phosphate74 o r T ~ ~ s - Hb uCf f e~r s~ or ~ o f decreasing c o n c e n t r a t i o n o f a s a l t (phosphate o r sulphate) i n water76 and combinations o f these g r a d i e n t s 7 4 y 7 5 a r e used, b u t a l i n e a r g r a d i e n t o f ethanol i n water77 and even o f urea i n a phosphate b u f f e r 7 8 have a l s o been employed. Various peptides, p r o t e i n s and enzymes have been chromatographed u s i n g t h i s technique. An example o f t h e s e p a r a t i o n o f O-amylase from b a r l e y meal i s shown i n F i g . 21.3. 21.6.
REVERSED-PHASE CHROMATOGRAPHY OF PEPTIDES AND POLYPEPTIDES
Recently, octadecyl and o c t y l s t a t i o n a r y phases c h e m i c a l l y bonded on a m i c r o p a r t i c u l a t e ( 5 o r 10
urn)
s i l i c a gel support have been used as column packing
m a t e r i a l s f o r reversed-phase chromatography. As i n hydrophobic i n t e r a c t i o n chromatography on o r g a n i c gels, t h e separation i s based on hydrophobic i n t e r a c t i o n s and t h e p e p t i d e s a r e separated on t h e b a s i s o f d i f f e r e n c e s i n t h e i r
373
100
200
400 Wml)
300
Fig. 21.3. F r a c t i o n a t i o n o f @-amylase from b a r l e y meal by hydrophobic i n t e r a c t i o n chromatography w i t h g r a d i e n t e l u t i o n . Column: Phenyl-Sepharose CL-4B, column K 16/20, bed volume 30 m l . Gradient: as i n d i c a t e d by broken l i n e . S o l v e n t s : A, 0.01 m o l / l sodium phosphate b u f f e r (pH 6.8), 25% s a t u r a t e d w i t h ammonium sulphate; B y 50% ( v / v ) e t h y l e n e g l y c o l i n water. Flow-rate: 0.417 ml/min. D e t e c t i o n : UV a t 280 nm. ,4280 = absorbance a t 280 nm; v = e l u t i o n volume. Sample: b a r l e y meal. B-Amylase enzymatic a c t i v i t y i s shown by t h e b r o k e n - l i n e peak. (Reproduced from r e f . 74 w i t h permission.) h y d r o p h o b i c i t i e s . T h i s technique has been a p p l i e d i n t h e high-speed chromatography o f a v a r i e t y o f peptides and s m a l l e r polypeptides. Gradients w i t h an i n c r e a s i n g c o n t e n t o f t h e organic s o l v e n t i n an aqueous m o b i l e phase a r e u s u a l l y used f o r e l u t i o n .
A l i n e a r g r a d i e n t o f a c e t o n i t r i l e i n water used f o r t h e chromatography o f c o l l a g e n peptides7’
i s an exception, because a c i d i c b u f f e r s o r aqueous s o l u t i o n s 80
o f a c i d s a r e u s u a l l y necessary f o r good separations. Gradients o f methanol acetoni t r i l e 30,81-87
,
o r propanol 6 8 y 1 0 2 i n a 0.1% aqueous s o l u t i o n o f phospho-
r i c 3 0 y 8 1 - 8 9 acetic8’,
f o r m i c l o 2 o r t r i f l u o r o a c e t i c acid68a87-89, a l i n e a r g r a d i e n t 90,91 o f a c e t o n i t r i l e i n a c i d i f i e d 0.155 m o l / l sodium c h l o r i d e s o l u t i o n (pH 2.1) , 101 o r isopropanol-2-methoxyethanol gradients o f acetoni trileg2-”, methanol loo 103-105 or i n phosphate b u f f e r s ( u s u a l l y a t pH 2-2.51, g r a d i e n t s o f a c e t o n i t r i l e methanol-acetic a c i d 106y107 i n 0.01 m o l / l ammonium a c e t a t e b u f f e r (pH 4-6), a l i n e a r g r a d i e n t o f methanol i n T r i s b u f f e r l o 8 , a l i n e a r g r a d i e n t o f n-propanol i n sodium c i t r a t e (pH 4.5)”’ i n p y r i d i n i u m formate b u f f e r ’ ”
I
and a l i n e a r g r a d i e n t o f n-propanol c o n c e n t r a t i o n have been used f o r e l u t i o n o f peptides.
The s e l e c t i v i t y o f reversed-phase separations o f peptides can sometimes be
‘,
improved by u s i n g i o n - p a i r f o r m a t i o n i n t h e mobile phase. 80th c a t i o n i c (such as t e t r a b u t y l ammoni um phosphate’
t r i e t h y l ammonium phosphate 13-’ l6and dodecyl-
amine117) and a n i o n i c (such as hexylsulphonate, decylsulphonate, p e n t a f l u o r o propanoic acid, h e p t a f l u o r o b u t y r i c a c i d and undecafluorocaproic a c i d ’ 18) i o n p a i r i n g reagents have been used f o r t h i s purpose. The i o n - p a i r i n g agent was References on p . 376.
374
u s u a l l y c o n t a i n e d i n t h e m o b i l e phase a t a c o n s t a n t c o n c e n t r a t i o n , w h i l e t h e c o n t e n t o f t h e o r g a n i c s o l v e n t ( a ~ e t o n i t r i l e l l ~ -methanol ~~~, o r i s o p r o p a n o l 'I6) changed 1 i n e a r l y d u r i n g g r a d i e n t e l u t i o n . Reversed-phase g r a d i e n t e l u t i o n chromatography on o c t y l - and o c t a d e c y l s i l i c a columns i s s u i t a b l e f o r t h e e f f i c i e n t and r a p i d a n a l y t i c a l and p r e p a r a t i v e mapping o f complex p e p t i d e m i x t u r e s and u s u a l l y y i e l d s b e t t e r s e p a r a t i o n s i n a s h o r t e r t i m e t h a n o t h e r l i q u i d chromatography t e c h n i q u e s . However, i t would be d i f f i c u l t t o a c h i e v e complete r e s o l u t i o n of a l l p e p t i d e s i n v e r y complex m i x t u r e s , such as i n t r y p t i c d i g e s t s o f l a r g e r p r o t e i n s , where i t c o u l d be necessary t o re-chromatograph a zone o f p a r t i a l l y r e s o l v e d p e p t i d e s i n a second, independent i s o c r a t i c run. To g i v e a few examples o f a p p l i c a t i o n s o f t h e reversed-phase chromatography
30
o f p e p t i d e s and small p r o t e i n s , we can mention chromatography o f p r o i n s u l i n , p e p t i d e s o f human f o e t a l g l o b i n 6 8 , n e ~ r o p e p t i d e s ~hypothalamic ~, 01 i g o p e p t i d e s 114 , enkephal i n peptides8' y 9 2 y 1 0 8 ,
argot ens in^"^,
parathyroid"
and a d r e n o c o r t i c o -
t r o p i c 9 2'1 l3hormones, pteroyl-oligo-y-L-g1utaminesg5, growth hormones8',
19-S
t h y l o g l o b u l i n 8 5 , o p i o i d peptides88 , o x y t o c i n , vasopressin, samostating6, t u f t s i n calcitonin112, a p o l i p r ~ t e i n s " ~ ,peptides o f r a t casein82y86, t r y p t i c d i g e s t o f
97
o f egg w h i t e l y s ~ z o m e ~o f~ sheep ~, 19-5 t h y r o g l o b u l i n and
haemoglobin8 4 y 1 0 3 y 1 0 4 ,
o f bovine thyrotrophin117. A h i g h - e f f i c i e n c y separation o f f o u r polypeptides i s shown i n F i g . 21.4 119
.
21.7.
AFFINITY CHROMATOGRAPHY OF PROTEINS
A f f i n i t y chromatography i s a h i g h l y s e l e c t i v e s e p a r a t i o n method t h a t a l l o w s t h e p u r i f i c a t i o n o f p r o t e i n s , p r o t e i n - b a s e d enzymes and hormones and o t h e r b i o p o l y m e r s on t h e b a s i s o f t h e i r b i o l o g i c a l f u n c t i o n s o r i n d i v i d u a l chemical s t r u c t u r e s . T h i s t e c h n i q u e makes i t p o s s i b l e t o remove l a r g e amounts o f cont a m i n a t i n g substances f r o m t h e s o l u t e s o f i n t e r e s t i n b i o l o g i c a l m a t e r i a l o r t o s e p a r a t e t h e a c t i v e f o r m f r o m t h e denatured forms o f t h e same substance. Theref o r e , t h e m e r i t s o f a f f i n i t y chromatography f o r b i o l o g i c a l r e s e a r c h a r e c o n s i d e r a b l e , and t h i s method has found wide acceptance s i n c e i t s f i r s t i n t r o d u c t i o n 120 about 15 y e a r s ago
.
I n p r i n c i p l e , t h e method i s based on t h e i n t e r a c t i o n s between a b i o s p e c i f i c l i g a n d c o v a l e n t l y bonded t o a s u i t a b l e s u p p o r t and t h e substance o f i n t e r e s t , t h e s p e c i f i c s t r u c t u r e o f which " f i t s " t h e s t r u c t u r e o f bonded ( i m m o b i l i z e d ) l i g a n d . F o r example, an enzyme bonded as t h e l i g a n d can be used f o r t h e p u r i f i c a t i o n o f a s u b s t r a t e analogue, i n h i b i t o r o r c o f a c t o r , o r an a n t i b o d y bonded as t h e l i g a n d may be s u i t a b l e f o r t h e p u r i f i c a t i o n o f an a n t i g e n o r o f a v i r u s , a n u c l e i c a c i d as t h e l i g a n d f o r p u r i f i c a t i o n o f n u c l e i c a t i d polymerase o r o f a b i n d i n g protein, etc.
375
2
I
0
1
8
I
16
t(min)
I
24
F i g . 21.4. S e p a r a t i o n o f f o u r p o l y p e p t i d e s by reversed-phase g r a d i e n t - e l u t i o n chromatography. Column: M i c r o Pak MCH, 150 x 5.4 mm I . D . G r a d i e n t : l i n e a r , 18-60% B i n 42 min (1% B/min). S o l v e n t s : A, triethylammonium phosphate b u f f e r (pH 2.2); B, a c e t o n i t r i l e . F l o w - r a t e : 1 ml/min. D e t e c t i o n : UV a t 210 nm. Peaks: 1 = bombesin; 2 = s o m a t o s t a t i n ; 3 = i n s u l i n ; 4 = glucagon. (Reproduced f r o m r e f . 119 w i t h p e r mission.) The use o f t h e method c o n s i s t s i n s e v e r a l c o n s e c u t i v e s t a g e s : ( a ) A s u i t a b l e b i o s p e c i f i c l i g a n d i s c o v a l e n t l y bonded t o a chromatographic bed m a t e r i a l ( m a t r i x ) , such as an agarose g e l (Sepharose 4B, Sepharose CL), p r e v i o u s l y a c t i v a t e d f o r e a s i e r c o v a l e n t attachment o f t h e l i g a n d . I t i s i m p o r t a n t t h a t the attached, "immobilized", l i g a n d r e t a i n s i t s s p e c i f i c b i n d i n g a c t i v i t y f o r t h e substance o f i n t e r e s t . ( b ) The g e l w i t h t h e " i m m o b i l i z e d " l i g a n d i s packed i n t o a column o f s u i t a b l e dimensions, t h e sample i s a p p l i e d , f r o m which substances c a p a b l e o f b i o s p e c i f i c i n t e r a c t i o n s w i t h t h e i m m o b i l i z e d l i g a n d become adsorbed on t h e column, w h i l e t h e non-sorbed i m p u r i t i e s a r e washed away f r o m t h e column w i t h a s t a r t i n g b u f f e r . References on p . 576.
376
( c ) E l u t i o n of t h e sorbed substances from t h e column u s i n g e l u e n t s t h a t decrease t h e a f f i n i t y f o r c e s between t h e substances and t h e immobilized l i g a n d . This i s u s u a l l y achieved by e i t h e r a stepwise o r a continuous change i n propert i e s of t h e e l u e n t and s e l e c t i v e o r n o n - s e l e c t i v e e l u e n t s can be employed, depending on t h e n a t u r e o f t h e b i n d i n g between t h e l i g a n d and s o l u t e s . Nons e l e c t i v e e l u t i o n methods i n v o l v e pH o r i o n i c s t r e n g t h g r a d i e n t s . A change ( u s u a l l y a decrease) i n pH d u r i n g g r a d i e n t e l u t i o n a l t e r s t h e degree o f i o n i z a t i o n of charged groups a t t h e b i n d i n g s i t e s and causes desorption. A g r a d i e n t o f i n c r e a s i n g i o n i c s t r e n g t h (e.g.,
o f sodium c h l o r i d e c o n c e n t r a t i o n ) a l s o leads
t o d e s o r p t i o n of sorbed s o l u t e s a t a c o n c e n t r a t i o n o f about 1 m o l / l . Urea and guanidine h y d r o c h l o r i d e a t h i g h c o n c e n t r a t i o n s a r e u s e f u l e l u e n t s , b u t they should be removed from t h e p u r i f i e d p r o t e i n s immediately a f t e r t h e e l u t i o n , otherwise they c o u l d cause d e n a t u r a t i o n o f p r o t e i n s . S e l e c t i v e ( a f f i n i t y ) e l u t i o n makes use o f 'an i n c r e a s i n g c o n c e n t r a t i o n g r a d i e n t o f an e l u t i n g agent t h a t e i t h e r competes f o r b i n d i n g t o t h e adsorbed substance o r f o r binding t o the ligand. To g i v e a few r a t h e r a r b i t r a r i l y chosen examples from t h e w e a l t h o f p u b l i s h e d i n f o r m a t i o n , we can mention chromatography o f phosphofructokinase on immobilized 121 Cibacron B l u e F 36-A u s i n g a l i n e a r g r a d i e n t (0-1.5 m o l / l ) o f ammonium s u l p h a t e , p u r i f i c a t i o n o f d i p e p t i d y l peptidase I V on t h e immobilized p e p t i d e Gly-Pro-NH(CH2)6-NH u s i n g a g r a d i e n t (0-0.2 m o l / l ) o f sodium c h l o r i d e 1 2 2 , f r a c t i o n a t i o n o f p r o t e o l y t i c enzymes t r y p s i n and chymotrypsin on immobilized a m i n o c a p r o y l p r o f l a v i n u s i n g a g r a d i e n t (0-0.08 m o l / l ) o f sodium chloride123, f r a c t i o n a t i o n o f Escker i c k i a coZi B aminoacyl-tRNA synthetases on immobilized hexylamine u s i n g a
g r a d i e n t (0-0.4 m o l / l ) o f potassium chloride124, p u r i f i c a t i o n o f a l k a l i n e phosphatase on immobilized t r i a z i n e dyes u s i n g a g r a d i e n t (0-1 m o l / l ) o f potassium c h l o r i d e 1 2 5 , f r a c t i o n a t i o n o f a complex m i x t u r e o f dehydrogenases on immobilized adenosine 5'-monophosphate u s i n g a pH g r a d i e n t (pH 6-10)Iz6, p u r i f i c a t i o n o f t r y p s i n and chymotrypsin on immobilized low-molecular-weight soybean protease i n h i b i t o r s u s i n g a pH g r a d i e n t (pH 8-4)127 and p u r i f i c a t i o n o f i n d o l ethylamine N-methyltransferase on immobilized S-adenosylhomocysteine128. I n most o f these a p p l i c a t i o n s , agarose g e l s were used as substrates. Comprehensive i n f o r m a t i o n about t h e v a r i o u s techniques and a p p l i c a t i o n s o f 129 a f f i n i t y chromatography can be found i n a monograph by Turkovd
.
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Chapter 2 1 PEPTIDES AND PROTEINS P r o t e i n s , t h e e s s e n t i a l components o f l i v i n g m a t t e r , a r e composed o f p o l y p e p t i d e c h a i n s . Enzymes and a number o f hormones a r e s t r u c t u r a l l y based on p r o t e i n s . A knowledge o f t h e amino a c i d sequence i n p e p t i d e s and i n p r o t e i n s i s v e r y i m p o r t a n t i n b i o c h e m i c a l r e s e a r c h . The number o f p u b l i s h e d a p p l i c a t i o n s o f l i q u i d chromatography t o t h e s e p a r a t i o n o f p e p t i d e s and p r o t e i n s i s enormous and perhaps outnumbers t h e sum o f l i q u i d chromatography a p p l i c a t i o n s t o t h e s e p a r a t i o n o f a l l t h e o t h e r compounds. I t would be i m p o s s i b l e h e r e t o g i v e a r e v i e w o f a l l s e p a r a t i o n methods i n which g r a d i e n t e l u t i o n has been a p p l i e d t o t h e chromatography o f p e p t i d e s and p r o t e i n s . T h e r e f o r e , a b r i e f survey o f some g e n e r a l l y a p p l i c a b l e methods employing t h e g r a d i e n t e l u t i o n t e c h n i q u e i s g i v e n and i l l u s t r a t e d , b y o n l y a few examples, t o g i v e a p i c t u r e o f what g r a d i e n t e l u t i o n l i q u i d column chromatography means i n s t u d i e s o f t h e s e compounds. A s y s t e m a t i c , comprehensive survey o f l i q u i d chromatography a p p l i c a t i o n s i n t h i s f i e l d may be f o u n d elsewhere (see, f o r example, r e f s . 1 - 3 ) . A p p l i c a t i o n s o f l i q u i d chromatography t o t h e p r e p a r a t i v e p u r i f i c a t i o n and f r a c t i o n a t i o n o f p r o t e i n s a r e i m p o r t a n t . High-performance l i q u i d chromatography (HPLC) has b r o u g h t new dimensions t o t h e a n a l y s i s o f p r o t e i n s , p e p t i d e s and amino a c i d s w i t h r e s p e c t t o speed o f a n a l y s i s , s e p a r a t i o n e f f i c i e n c y and s e n s i t i v i t y , which makes i t p o s s i b l e t o a c h i e v e m i c r o - s c a l e s e p a r a t i o n s o f complex m i x t u r e s of p e p t i d e s i n s m a l l amounts o f samples i n l e s s t h a n 1 h, i n c o n t r a s t 133 t o e a r l i e r e l a b o r a t e and time-consuming methods g i v i n g l o w y i e l d s o f m a t e r i a l
.
To d e t e r m i n e t h e amino a c i d c o m p o s i t i o n o f p r o t e i n s and p e p t i d e s , i t i s necessary f i r s t t o s u b j e c t t h e s e compounds t o t o t a l o r s e q u e n t i a l h y d r o l y s i s . I n t o t a l h y d r o l y s a t e s t h e r a t i o o f t h e i n d i v i d u a l amino a c i d s can be determined, b u t s e q u e n t i a l h y d r o l y s i s a l l o w s t h e d e t e r m i n a t i o n o f t h e sequence o f amino a c i d s i n p e p t i d e c h a i n s . Edman d e g r a d a t i o n i s a n o n - d e s t r u c t i v e ,
subtractive
method o f p r o t e i n a n a l y s i s u s i n g r e p e a t e d d e g r a d a t i o n o f p e p t i d e s o r p r o t e i n s i n three-step cycles. I n the f i r s t step, t h e peptide o r p r o t e i n reacts w i t h phenyl i s o t h i o c y a n a t e t o g i v e t h e phenyl t h i o c a r b a m y l (PTC) d e r i v a t i v e . I n t h e second step, t h e d e r i v a t i z e d N - t e r m i n a l amino ac d i s h y d r o l y s e d f r o m t h e remainder o f t h e p r o t e i n o r p e p t i d e and i t s 2-an l i n o - 5 - t h i a z o l i n e d e r i v a t i v e i s formed and c o n v e r t e d i n t o t h e s t a b l e 3-phenyl 2 - t h i o h y d a n t o i n (PTH) d e r i v a t i v e References on p . 376.
366 i n t h e t h i r d step. Because o n l y t h e N-terminal amino a c i d i s cleaved i n t h i s procedure and t h e remainder o f t h e p e p t i d e o r p r o t e i n remains i n t a c t , t h e whole procedure can be repeated as many times as necessary t o f i n d t h e amino a c i d sequence. L i q u i d chromatography i s v e r y s u i t a b l e f o r combination w i t h Edman degradation as a r a p i d and r e l i a b l e a n a l y t i c a l technique t h a t makes i t p o s s i b l e t o p u r i f y and t o check t h e p u r i t y of o r i g i n a l p r o t e i n s o r p e p t i d e s and o f PTC d e r i v a t i v e s formed i n t h e f i r s t s t e p of Edman degradation, t o separate, i d e n t i f y and quant i t a t e PTH-amino a c i d d e r i v a t i v e s formed i n each t h r e e - s t e p Edman degradation cycle. I n a l l o f these l i q u i d chromatography a p p l i c a t i o n s g r a d i e n t e l u t i o n , e i t h e r continuous o r stepwise, may be used. I n t h e subsequent d e t e r m i n a t i o n o f t h e amino a c i d sequence i n p r o t e i n and p e p t i d e hydrolysates, t h e methods f o r amino a c i d a n a l y s i s a r e a p p l i e d ; t h e a p p l i c a t i o n s o f t h e g r a d i e n t e l u t i o n technique f o r t h i s purpose were t r e a t e d i n Chapter 20. P r i o r t o t h e d e t e r m i n a t i o n o f t h e amino a c i d r a t i o and/or sequence o r f o r o t h e r biochemical research, i t i s necessary t o p u r i f y and separate p r o t e i n s and peptides i n mixtures, and l i q u i d column chromatography i s u n i q u e l y s u i t e d f o r t h i s purpose. However, t h e s p e c i f i c f r a g i l e c h a r a c t e r o f t h e biopolymers should be borne i n mind when s e l e c t i n g t h e chromatographic c o n d i t i o n s , i n o r d e r t o avoid the r i s k o f possible denaturation o f proteins. This r i s k i s p a r t i c u l a r l y important w i t h enzymes, where t h e i r s p e c i f i c topography, which g i v e s r i s e t o t h e i r p a r t i c u l a r a c t i v i t i e s , may become l o s t by s t r u c t u r a l changes when t h e
.
enzyme i s brought i n t o c o n t a c t w i t h an u n s u i t a b l e column packing m a t e r i a l 3 I n numerous e a r l i e r a p p l i c a t i o n s o f l i q u i d chromatography t o t h e p u r i f i c a t i o n and f r a c t i o n a t i o n o f peptides and p r o t e i n s , f r a c t i o n s o f t h e e l u a t e were c o l l e c t e d and t h e amount o f compounds e l u t e d was determined u s i n g spectrophotom e t r i c methods, measurement o f t h e r a d i o a c t i v i t y o f l a b e l l e d compounds, measurement o f enzymatic a c t i v i t y , e t c . I n more r e c e n t a p p l i c a t i o n s , m a i n l y those u s i n g e f f i c i e n t columns, t h e e l u a t e s a r e monitored c o n t i n u o u s l y . The d e t e c t i o n o f aromatic peptides employs U V a b s o r p t i o n a t 280 nm. U V detect i o n a t 254 nm has o f t e n been u t i l i z e d i n t h e chromatography o f p r o t e i n s , hormones and enzymes based on p r o t e i n s . Non-aromatic peptides were detected i n t h e U V r e g i o n between 200 and 210 nm. Enzymes i n t h e e l u a t e can be detected by continuous measurement o f t h e i r enzymatic a c t i v i t y and l a b e l l e d compounds by continuous m o n i t o r i n g o f r a d i o a c t i v i t y , Post-column d e r i v a t i z a t i o n i s o f t e n used f o r t h e d e t e c t i o n o f p r o t e i n s and peptides. D e r i v a t i v e s w i t h o-phthalaldehyde o r o t h e r reagents a r e d e t e c t e d i n t h e column e f f l u e n t s employing flow-through r e a c t o r s connected w i t h f l u o r i m e t r i c d e t e c t o r s o r w i t h photometric d e t e c t o r s o p e r a t i n g i n t h e U V o r v i s i b l e region.
36 7 21.1.
ION-EXCHANGE CHROMATOGRAPHY
Ion-exchange chromatography has been most f r e q u e n t l y used i n t h e chromatography o f p e p t i d e s and p r o t e i n s . The s e p a r a t i o n o f p e p t i d e s and p r o t e i n s by ion-exchange chromatography i s based on e l e c t r o s t a t i c i n t e r a c t i o n s between t h e charged exchange groups o f t h e exchanger and t h e o p p o s i t e charge s i t e s o f t h e p e p t i d e o r p r o t e i n . The amphoteric c h a r a c t e r o f t h e s e compounds a l l o w s b o t h c a t i o n and a n i o n exchangers t o be used. A t t h e i s o e l e c t r i c p o i n t , t h e p o s i t i v e and n e g a t i v e charges o f t h e s o l u t e a r e i n e q u i l i b r i u m . A t a pH l o w e r t h a n t h a t c o r r e s p o n d i n g t o t h e i s o e l e c t r i c p o i n t , t h e p r o t e i n o r p e p t i d e i s r e t a i n e d on a c a t i o n exchanger owing t o t h e e l e c t r o s t a t i c f o r c e s , whereas i t i s u n r e t a i n e d on an a n i o n exchanger. The o p p o s i t e h o l d s t r u e a t a pH above t h a t c o r r e s p o n d i n g t o t h e i s o e l e c t r i c p o i n t . However, t h e use o f a n i o n exchangers i s o f t e n p r e f e r r e d owing t o t h e b e t t e r s t a b i l i t y o f p r o t e i n s i n t h e i r a n i o n i c forms. To improve t h e p o s s i b i l i t i e s o f p e n e t r a t i o n o f l a r g e p r o t e i n m o l e c u l e s i n t o t h e i n t e r i o r o f t h e p a r t i c l e s , exchangers w i t h wide pores a r e s e l e c t e d . Because t h e r i g i d i t y o f exchangers w i t h an o r g a n i c m a t r i x decreases and t h e i r s w e l l i n g i n c r e a s e s w i t h i n c r e a s i n g p o r e s i z e s , which i s o b v i o u s l y disadvantageous f r o m t h e p o i n t o f view o f column bed s t a b i l i t y and maximum t o l e r a b l e f l o w - r a t e s , narrow-pore i o n exchangers a r e sometimes p r e f e r r e d f o r t h e chromatography o f very l a r g e proteins. P o l y i o n s such as p e p t i d e s and p r o t e i n s a r e n o r m a l l y v e r y f i r m l y a t t a c h e d t o i o n exchangers by many i o n i c groups i n t h e i r molecules. To a c h i e v e t h e i r e l u t i o n , i t i s necessary t o employ a b u f f e r w i t h pH a b o u t 1 u n i t above ( i n c o n n e c t i o n
w i t h c a t i o n exchangers) o r below ( w i t h a n i o n exchangers) t h e i s o e l e c t r i c p o i n t o f t h e p r o t e i n o r p e p t i d e . T h e r e f o r e , optimum c o n d i t i o n s o f e l u t i o n s h o u l d be e s t a b l i s h e d f o r each compound and i t i s g e n e r a l l y necessary t o change t h e c o m p o s i t i o n o f t h e m o b i l e phase i n t h e c o u r s e o f e l u t i o n . Stepwise e l u t i o n can be used f o r t h i s purpose; however, a l l substances r e l e a s e d by a change o f b u f f e r c o m p o s i t i o n a r e e l u t e d t o g e t h e r , u n f r a c t i o n a t e d , u s u a l l y as peaks w i t h s h a r p f r o n t s and pronounced t a i l i n g . F a l s e peaks may o c c u r i f t h e b u f f e r c o m p o s i t i o n i s changed b e f o r e t h e complete e l u t i o n o f t h e t a i l i n g peak has been achieved. T h i s drawback i s a v o i d e d when u s i n g c o n t i n u o u s g r a d i e n t s f o r e l u t i o n , w h i c h y i e l d s symmetrical peaks, b e t t e r r e s o l v e d f r o m each o t h e r t h a n i n s t e p w i s e elution. During gradient e l u t i o n , t h e solutes a r e e l u t e d i n order o f t h e i r i s o e l e c t r i c p o i n t s . I n a g r a d i e n t o f i n c r e a s i n g i o n i c s t r e n g t h a t a pH c l o s e t o t h e i s o e l e c t r i c p o i n t s o f s o l u t e s , t h e s a l t i n t h e e l u e n t competes w i t h t h e s o l u t e s f o r t h e exchange groups o f t h e exchanger and t h i s competing e f f e c t i n c r e a s e s w i t h i n c r e a s i n g i o n i c s t r e n g t h o f t h e m o b i l e phase. References on p . 376.
368 The i o n i c s t r e n g t h can be changed b y changing t h e c o n c e n t r a t i o n o f t h e b u f f e r o r , more f r e q u e n t l y , by i n c r e a s i n g t h e c o n c e n t r a t i o n o f a n e u t r a l s a l t . I d e a l l y , t h e i o n i c s t r e n g t h o f t h e s t a r t i n g b u f f e r s h o u l d be low. However, b u f f e r s w i t h v e r y l o w i o n i c s t r e n g t h s have low b u f f e r i n g c a p a c i t i e s and l a r g e changes i n i o n i c s t r e n g t h may cause l a r g e changes i n t h e volumes o f s w o l l e n exchangers o f c e r t a i n t y p e s and, t h e r e f o r e , b u f f e r s w i t h v e r y l o w i o n i c s t r e n g t h s s h o u l d b e avoided. I f a pH g r a d i e n t i s a p p l i e d f o r chromatography o f p r o t e i n s o r p e p t i d e s on a cation-exchange column, t h e o r i g i n a l pH i s s e l e c t e d below t h a t c o r r e s p o n d i n g t o t h e l o w e s t i s o e l e c t r i c p o i n t o f t h e compound i n t h e sample m i x t u r e and t h e n i t i s i n c r e a s e d w i t h t i m e t o a c h i e v e t h e e l u t i o n and s e p a r a t i o n . I n c o n t r a s t ,
t h e o r i g i n a l pH above t h e i s o e l e c t r i c p o i n t s o f s o l u t e s i s s e l e c t e d i n a n i o n exchange chromatography and i t i s decreased d u r i n g t h e e l u t i o n .
resin^^-^"'
C a t i o n - o r an on-exchange polystyrene-divinylbenzene , ion exchangers based on m o d i f i e d c e l l u l ~ s e ~ -d ~e x~t r,a n (Sephadex) 24-32,130, agarose 32,33 o r g l y c o l m e t h a c r y l a t e (Spheron) g e l s 34-39 and i n o r g a n i c i o n (Sepharose) exchangers, such as h y d r o x y a p a t i t e w i t h anion-exchange
proper tie^^'-^*,
have
been w i d e l y appl ed f o r t h e p u r i f i c a t i o n and f r a c t i o n a t i o n o f p e p t i d e s and p r o t e i n s b y 1 i q u d chromatography. Most c l a s s i c a l i o n exchangers do n o t w i t h s t a n d e l e v a t e d p r e s s u r e s and t h e r e f o r e r a p i d analyses a t e l e v a t e d f l o w - r a t e s o f t h e m o b i l e phase a r e n o t p o s s i b l e . Spheron exchangers and some exchangers based on s t y r e n e - d i v i n y l b e n z e n e copolymers a r e e x c e p t i o n s f r o m t h i s r u l e . However, p r o t e i n s may become denatured by v i r t u e o f hydrophobic i n t e r a c t i o n s w i t h t h e p o l y s t y r e n e m a t r i x . R e a l l y r a p i d analyses o f p r o t e i n s and p e p t i d e s have become p o s s i b l e o n l y w i t h i o n exchangers based on m i c r o p a r t i c u l a t e i n o r g a n i c s u p p o r t s w i t h c h e m i c a l l y bonded ion-exchange f u n c t i o n a l groups, which a r e s u f f i c i e n t l y r i g i d and f o r m s t a b l e column beds a t h i g h p r e s s u r e s . An o r g a n i c s u r f a c e l a y e r bonded on t h e i n o r g a n i c s u p p o r t i s u s e f u l f o r p r o t e c t i n g p r o t e i n s f r o m d i r e c t c o n t a c t w i t h t h e i n o r g a n i c surface and c o n t r i b u t e s t o t h e chromatographic p a r t i t i o n i n g process i n a s i m i l a r manner t o ion-exchange r e s i n s o r m o d i f i e d g e l s . An i m p o r t a n t advance was t h e i n t r o d u c t i o n o f column p a c k i n g m a t e r i a l s p r e p a r e d by chemical bonding o f g l y c e r y l p r o p y l s i l a n e on t h e s u r f a c e o f c o n t r o l l e d p o r o s i t y glass. The p r o d u c t r e t a i n s i t s t e r m i n a l epoxy group, which can be s u b j e c t e d t o r e a c t i o n w i t h a n u c l e o p h i l i c reagent t o g i v e t h e required chemically modified m a t e r i a l . These m a t e r i a l s have been marketed under t h e name Glycophase/CPG. I o n exchangers o f t h i s t y p e (DEAE-, QAE-, SP- o r SM-Glycophase/CPG) 43-51 o r i o n exchangers c h e m i c a l l y bonded on a s i l i c a g e l s u p p o r t 52-55 made p o s s i b l e h i g h speed s e p a r a t i o n s o f p r o t e i n s and p e p t i d e s . Because ion-exchange chromatography can r e s o l v e p r o t e i n s w i t h s m a l l charge d i f f e r e n c e s , i t i s p o s s i b l e t o s e p a r a t e
369 isoenzymes t h a t have n e a r l y i d e n t i c a l amino a c i d sequences i n t h e s e h i g h l y s e l e c t i v e and e f f i c i e n t chromatographic systems. C o n c e n t r a t i o n ( i o n i c s t r e n g t h ) o r pH g r a d i e n t s i n m o b i l e phases a r e u s u a l l y formed f r o m s o l v e n t s c o n t a i n i n g d i f f e r e n t c o n c e n t r a t i o n s o f phosphate, a c e t a t e o r p y r i d i n e - a c e t a t e b u f f e r s i n water. J u s t t o o b t a i n an i n s i g h t i n t o t h e v a r i e t y o f a p p l i c a t i o n s p o s s i b l e , we can mention a few examples, such as chromatography o f serum p r o t e i n s on an a n i o n exchange r e s i n u s i n g a c o n c e n t r a t i o n g r a d i e n t o f a c e t a t e b u f f e r 8 ; t h e d e t e r m i n a t i o n o f reduced and o x i d i z e d g l u t a t h i o n e i n wheat f l o u r s and doughs on a c a t i o n exchange r e s i n w i t h a g r a d i e n t o f h y d r o c h l o r i c a c i d c o n c e n t r a t i o n 7 ; t h e s e p a r a t i o n o f p r o t e i n s i n human and animal s e r a by chromatography on DEAE-cellulose a n i o n exchanger u s i n g c ~ n c e n t r a t i o n lo~r pH12 g r a d i e n t s i n phosphate b u f f e r s , c o n c e n t r a t i o n gradients i n T r i s buffers16y22y23y131
o r gradients o f increasing concentration
o f sodium c h l o r i d e i n water2' ; t h e f r a c t i o n a t i o n o f t y r o s i n e - r i c h p r o t e i n s f r o m wool on a Q A E - c e l l u l o s e a n i o n exchanger u s i n g a g r a d i e n t o f i o n i c s t r e n g t h i n a l k a l i n e B-alanine buffer";
and t h e s e p a r a t i o n and p u r i f i c a t i o n o f t h e enzymes
8-glucan h y d r o l a s e 1 5 and B-N-acetyl -D-hexosaminidase19 on DEAE-cell u l o s e u s i n g an i o n i c s t r e n g t h ( c o n c e n t r a t i o n o f a l k a l i m e t a l c h l o r i d e ) g r a d i e n t i n a phosphate b u f f e r . We can f u r t h e r m e n t i o n t h e chromatography o f i n s u l i n and i t s d e r i v a t i v e s on a DEAE-Sephadex a n i o n exchanger u s i n g a c o n c e n t r a t i o n g r a d i e n t o f sodium c h l o r i d e i n a Tris-HC1 b u f f e r c o n t a i n i n g 7 m o l / l urea26; t h e chromatography o f t h y m o p o i e t i n s on QAE-Sephadex u s i n g a c o n c e n t r a t i o n g r a d i e n t of potassium c h l o r i d e 1 3 2 ; t h e f r a c t i o n a t i o n o f whey p r o t e i n s and c a s e i n i n cows' m i l k on t h e same m a t e r i a l u s i n g a c o n c e n t r a t i o n g r a d i e n t o f sodium c h l o r i d e i n a phosphate b u f f e r 2 4 ; t h e chromatography o f enzymes r i b o n u c l e a s e s on a CM-Sephadex c a t i o n exchanger u s i n g combined pH and i o n i c s t r e n g t h g r a d i e n t s i n a phosphate b u f f e r 3 1 ; t h e chromatography o f a - b u n g a r o t o x i n and o t h e r components o f snake venom on t h e same column p a c k i n g w i t h a l i n e a r c o n c e n t r a t i o n g r a d i e n t o f ammonium a c e t a t e i n w a t e r
(0.05-1 m ~ l / l ) ' ~and ~ ; t h e f r a c t i o n a t i o n o f human serum p r o t e i n s by a n i o n exchange chromatography on DEAE-Sepharose C1-6B u s i n g a l i n e a r g r a d i e n t o f i n c r e a s i n g sodium c h l o r i d e c o n c e n t r a t i o n i n 0.05 m o l / l Tris-HC1 b u f f e r a t pH 8.6
33
On Spheron i o n exchangers, s i m i l a r t y p e s o f compounds can be chromatographed as on c e l l u l o s e o r p o l y d e x t r a n exchangers, b u t Spheron m a t e r i a l s have t h e advantages o f b e t t e r mechanical r i g i d i t y and s t a b i l i t y a t h i g h e r p r e s s u r e s , which a l l o w s more r a p i d s e p a r a t i o n s t o be a c h i e v e d a t h i g h e r f l o w - r a t e s o f t h e m o b i l e phase. An example o f g r a d i e n t e l u t i o n chromatography o f human plasma on DEAE-Spheron column i s shown i n F i g . 21.1.
The e l u t i o n p r o f i l e h e r e i s s i m i l a r t o t h a t o b t a i n e d
on D E A E - c e l l ~ l o s e ~ O ~n. h y d r o x y a p a t i t e columns, f o r example, f r a c t i o n a t i o n o f myos in40y41 and
his tone^^^ was
achieved u s i n g i o n i c s t r e n g t h g r a d i e n t s i n phos-
p h a t e b u f f e r s . By chromatography on DEAE-Glycophase/CPG u s i n g 1 i n e a r c o n c e n t r a t i o n
References on p . 3 7 6 .
.
370
30
1.0
60
90
t(min) 120
[roo
70C
A,,,
100
0.6
0.2 10
40
70
F.N.
t
Y o0
%A
loo
F i g . 21.1. P r o t e i n mapping o f human plasma by ion-exchange l i q u i d chromatography on a m o d i f i e d diethylaminoethyl-Spheron anion exchanger. Column: DEAE-Spheron, 25-32 pm, 190 x 8 mm I.D. Gradient: m u l t i - l i n e a r , as i n d i c a t e d by t h e t h i n f u l l l i n e , from 0 t o 100% B i n A and then from 0 t o 100% C i n B. B u f f e r s : S o l v e n t A, 0.025 m o l / l phosphoric a c i d - T r i s (pH 8 . 5 ) ; s o l v e n t B, 0.5 m o l / l phosphoric a c i d T r i s (pH 3.5); s o l v e n t C, s o l v e n t B w i t h 1.0 m o l / l KC1. Flow-rate: 3.75 ml/min. F r a c t i o n s c o l l e c t e d a t 1.5-min i n t e r v a l s . F.N. = f r a c t i o n number; 4254 = absorbance a t 254 nm. Sample: 2 m l o f human plasma, c e n t r i f u g e d b e f o r e a p p l i c a t i o n . (Reproduced from r e f . 34 w i t h permission.) g r a d i e n t s o f sodium c h l o r i d e i n a c e t a t e o r Tris-HC1 b u f f e r s (pH 8 ) , i t was p o s s i b l e t o separate isoenzymes o f l a c t a t e dehydrogenase, hexokinase, etc.,
in
25-40 min45-50. Human serum p r o t e i n s and r a t kidney and r a t l i v e r isoenzymes c o u l d be r a p i d l y separated by anion-exchange chromatography on a P E I 6-LiChrospher S i 500-column ( a p e l l i c u l a r anion exchanger on a s i l i c a gel support) u s i n g l i n e a r
g r a d i e n t s o f sodium a c e t a t e c o n c e n t r a t i o n i n 0.02 m o l / l T r i s - a c e t a t e b u f f e r
21.2.
CHROMATOFOCUSING
pH Gradients may be generated i n an ion-exchange column i t s e l f , when i t i s o r i g i n a l l y e q u i l i b r a t e d w i t h a b u f f e r w i t h a pH d i f f e r e n t t o t h a t o f t h e b u f f e r used f o r e l u t i o n . Using a s p e c i a l b l e n d o f b u f f e r s w i t h even b u f f e r i n g c a p a c i t i e s over a wide pH range and s p e c i a l i o n exchangers, a smooth continuous pH g r a d i e n t ,,56-59
may be generated on t h e column. T h i s technique, s o - c a l l e d "chromatofocusing
i s t r e a t e d i n more d e t a i l i n P a r t 111. Very s i m i l a r i n p r i n c i p l e i s s o - c a l l e d "ampholyte-displacement
chromatography"60. An example o f t h e s e p a r a t i o n o f
s o l u b l e p r o t e i n s e x t r a c t e d from e l k muscle u s i n g t h e chromatofocusing technique on agarose-derived i o n exchangers ( P o l y b u f f e r exchangers) i s shown i n F i g . 21.2
59
.
371
0.5 A280
0.4
9 PH
0.3
8
0.2
7
0.1
0
50
100
150
200
. 6250
V(ml)
F i g . 21.2. F r a c t i o n a t i o n o f s o l u b l e p r o t e i n s e x t r a c t e d f r o m e l k muscle by c h r o matofocusing. Column: Pharmacia C 10/40; packed w i t h p o l y b u f f e r exchanger, PBE 94, bed h e i g h t = 45 mm. Column e q u i l i b r a t i o n b u f f e r , 0.025 m o l / l ethanol-amineaHC1 (pH 9.4); e l u t i o n b u f f e r , 0.0075 mmol/pH u n i t / m l P o l y b u f f e r 96 (pH 6 ) . Column s e l f - g e n e r a t e d pH g r a d i e n t i s i n d i c a t e d by t h e f u l l l i n e . F l o w - r a t e : 20 cm/h. D e t e c t i o n : UV a t 280 nm. A 0 absorbance a t 280 nm; V = e l u t i o n volume. Sample: 5 m l o f s u p e r n a t a n t f r o m ef! m i a t homogenate. (Reproduced f r o m r e f . 59 w i t h permi s s i o n . ) 21.3.
CHROMATOGRAPHY ON PDLYAMIDE GELS
Polyamide g e l s w i t h amide groups can behave as weak a n i o n exchangers and bond p e p t i d e s and p r o t e i n s by e l e c t r o s t a t i c f o r c e s . T h e r e f o r e , t h e s e compounds can be chromatographed on polyamide g e l s , such as Bio-Gel P, u s i n g e l u t i o n w i t h pH o r i o n i c s t r e n g t h g r a d i e n t s i n a c e t a t e , phosphate, Tris-HC1 o r o t h e r b u f f e r s . T h i s t e c h n i q u e was a p p l i e d , f o r example, t o s e p a r a t i o n s o f m e t h i o n y l p e p t i d e s
61
,
t h e chromatography o f i o d i n a t e d L - t y r o s y l - L - t y r o s i n e peptides6’ and t h e r e s o l u t i o n 63 o f venom p r o t e i n s
.
21.4.
CHROMATOGRAPHY ON POLAR ADSORBENTS AND ON POLAR BONDED PHASES
Chromatography on p o l a r adsorbents i s r e l a t i v e l y r a r e l y used f o r s e p a r a t i o n s o f p r o t e i n s and p e p t i d e s . UV-absorbing d e r i v a t i v e s o f p e p t i d e s ( p h e n y l t h i o h y d a n t o i n and benzoyl d e r i v a t i v e s ) were chromatographed on s i l i c a g e l u s i n g e l u t i o n w i t h a c o n t i n u o u s g r a d i e n t o f e t h a n o l i n c h l ~ r o f o r m ~p~r o, p a n o l , methanol and d i c h l o r o m e t h a n e i n n - h e ~ a n eo~r ~s t e p w i s e g r a d i e n t s o f anhydrous e t h a n o l and a c e t i c a c i d i n dichloromethane66. UV d e t e c t i o n a t 254 nm i s s u i t a b l e w i t h t h e s e systems. P e p t i d e s o f human f o e t a l g l o b i n and o f m u r i n e l a a n t i g e n s were s e p a r a t e d by chromatography on a n i t r i l e phase bonded on s i l i c a (Zorbax CN) u s i n g a c o n t i n u o u s g r a d i e n t o f o r g a n i c s o l v e n t i n aqueous a c i d i c medium (acetone i n phosphate b u f f 68 e r , pH o r propanol i n 0.1% t r i f l u o r o a c e t i c a c i d ) f o r e l u t i o n .
d7,
References on p . 376.
372 21.5.
HYDROPHOBIC INTERACTION CHROMATOGRAPHY
It has been observed t h a t i n chromatography on g e l s i n aqueous media, p r o t e i n s
and peptides a r e e l u t e d i n disagreement w i t h t h e i r expected e l u t i o n o r d e r acc o r d i n g t o t h e i r molecular s i z e s and t h a t t h e r e t e n t i o n i s i n f l u e n c e d by t h e mobile phase composition. Therefore', i t i s obvious t h a t , i n a d d i t i o n t o a p o s s i b l e s t e r i c e x c l u s i o n mechanism, o t h e r i n t e r a c t i o n s c o n t r o l t h e chromatographic process I t has been e s t a b l i s h e d t h a t these i n t e r a c t i o n s a r e hydrophobic, i.e.,
t h a t they
o r i g i n a t e i n a t t r a c t i o n f o r c e s between water molecules, by v i r t u e o f which t h e molecules of biopolymers a r e excluded from t h e m o b i l e phase on t o t h e gel surface. T h i s e f f e c t i s o f a s i m i l a r n a t u r e as i n reversed-phase chromatography on chemic a l l y bonded non-polar a l k y l o r a r y l phases. Peptides, p r o t e i n s and o t h e r b i o polymers c o u l d be separated on non-polar o r s l i g h t l y p o l a r o r g a n i c g e l s ( o r on m a t e r i a l s w i t h a c h e m i c a l l y bonded o r g a n i c l a y e r on an i n o r g a n i c support, such as G l y ~ o p h a s e - G / C P G ) ~u~s i n g t h i s s e p a r a t i o n mechanism and t h e corresponding chromatographic technique has been c a l l ed graphy" 6 9 y 7 0 .
'I
hydrophobi c i n t e r a c t i o n chromato-
As i n chromatography on c h e m i c a l l y bonded non-polar phases, r e t e n -
t i o n increases w i t h i n c r e a s i n g c o n c e n t r a t i o n o f a s a l t and decreases w i t h i n c r e a s i n g c o n c e n t r a t i o n o f an o r g a n i c s o l v e n t i n t h e mobile phase. Gels cont a i n i n g e t h e r e a l bonds and hydroxy o r e s t e r groups, such as unmodified Spheron gel72, can be used f o r t h e hydrophobic i n t e r a c t i o n chromatography o f p r o t e i n s and peptides, b u t agarose g e l s w i t h coupled hydrophobic groups, such as o c t y l , phenyl o r benzyl, have been s p e c i a l l y developed f o r t h i s purpose. The o r g a n i c s o l v e n t f o r t h e mobile phase g r a d i e n t should be s e l e c t e d w i t h c a r e and t h e s o l u b i l i t y o f p r o t e i n s i n t h e mobile phase should be borne i n mind. Most o f t e n , 73 g r a d i e n t s o f i n c r e a s i n g c o n c e n t r a t i o n o f e t h y l e n e g l y c o l (0-50%) i n a c e t a t e , phosphate74 o r T ~ ~ s - Hb uCf f e~r s~ or ~ o f decreasing c o n c e n t r a t i o n o f a s a l t (phosphate o r sulphate) i n water76 and combinations o f these g r a d i e n t s 7 4 y 7 5 a r e used, b u t a l i n e a r g r a d i e n t o f ethanol i n water77 and even o f urea i n a phosphate b u f f e r 7 8 have a l s o been employed. Various peptides, p r o t e i n s and enzymes have been chromatographed u s i n g t h i s technique. An example o f t h e s e p a r a t i o n o f O-amylase from b a r l e y meal i s shown i n F i g . 21.3. 21.6.
REVERSED-PHASE CHROMATOGRAPHY OF PEPTIDES AND POLYPEPTIDES
Recently, octadecyl and o c t y l s t a t i o n a r y phases c h e m i c a l l y bonded on a m i c r o p a r t i c u l a t e ( 5 o r 10
urn)
s i l i c a gel support have been used as column packing
m a t e r i a l s f o r reversed-phase chromatography. As i n hydrophobic i n t e r a c t i o n chromatography on o r g a n i c gels, t h e separation i s based on hydrophobic i n t e r a c t i o n s and t h e p e p t i d e s a r e separated on t h e b a s i s o f d i f f e r e n c e s i n t h e i r
373
100
200
400 Wml)
300
Fig. 21.3. F r a c t i o n a t i o n o f @-amylase from b a r l e y meal by hydrophobic i n t e r a c t i o n chromatography w i t h g r a d i e n t e l u t i o n . Column: Phenyl-Sepharose CL-4B, column K 16/20, bed volume 30 m l . Gradient: as i n d i c a t e d by broken l i n e . S o l v e n t s : A, 0.01 m o l / l sodium phosphate b u f f e r (pH 6.8), 25% s a t u r a t e d w i t h ammonium sulphate; B y 50% ( v / v ) e t h y l e n e g l y c o l i n water. Flow-rate: 0.417 ml/min. D e t e c t i o n : UV a t 280 nm. ,4280 = absorbance a t 280 nm; v = e l u t i o n volume. Sample: b a r l e y meal. B-Amylase enzymatic a c t i v i t y i s shown by t h e b r o k e n - l i n e peak. (Reproduced from r e f . 74 w i t h permission.) h y d r o p h o b i c i t i e s . T h i s technique has been a p p l i e d i n t h e high-speed chromatography o f a v a r i e t y o f peptides and s m a l l e r polypeptides. Gradients w i t h an i n c r e a s i n g c o n t e n t o f t h e organic s o l v e n t i n an aqueous m o b i l e phase a r e u s u a l l y used f o r e l u t i o n .
A l i n e a r g r a d i e n t o f a c e t o n i t r i l e i n water used f o r t h e chromatography o f c o l l a g e n peptides7’
i s an exception, because a c i d i c b u f f e r s o r aqueous s o l u t i o n s 80
o f a c i d s a r e u s u a l l y necessary f o r good separations. Gradients o f methanol acetoni t r i l e 30,81-87
,
o r propanol 6 8 y 1 0 2 i n a 0.1% aqueous s o l u t i o n o f phospho-
r i c 3 0 y 8 1 - 8 9 acetic8’,
f o r m i c l o 2 o r t r i f l u o r o a c e t i c acid68a87-89, a l i n e a r g r a d i e n t 90,91 o f a c e t o n i t r i l e i n a c i d i f i e d 0.155 m o l / l sodium c h l o r i d e s o l u t i o n (pH 2.1) , 101 o r isopropanol-2-methoxyethanol gradients o f acetoni trileg2-”, methanol loo 103-105 or i n phosphate b u f f e r s ( u s u a l l y a t pH 2-2.51, g r a d i e n t s o f a c e t o n i t r i l e methanol-acetic a c i d 106y107 i n 0.01 m o l / l ammonium a c e t a t e b u f f e r (pH 4-6), a l i n e a r g r a d i e n t o f methanol i n T r i s b u f f e r l o 8 , a l i n e a r g r a d i e n t o f n-propanol i n sodium c i t r a t e (pH 4.5)”’ i n p y r i d i n i u m formate b u f f e r ’ ”
I
and a l i n e a r g r a d i e n t o f n-propanol c o n c e n t r a t i o n have been used f o r e l u t i o n o f peptides.
The s e l e c t i v i t y o f reversed-phase separations o f peptides can sometimes be
‘,
improved by u s i n g i o n - p a i r f o r m a t i o n i n t h e mobile phase. 80th c a t i o n i c (such as t e t r a b u t y l ammoni um phosphate’
t r i e t h y l ammonium phosphate 13-’ l6and dodecyl-
amine117) and a n i o n i c (such as hexylsulphonate, decylsulphonate, p e n t a f l u o r o propanoic acid, h e p t a f l u o r o b u t y r i c a c i d and undecafluorocaproic a c i d ’ 18) i o n p a i r i n g reagents have been used f o r t h i s purpose. The i o n - p a i r i n g agent was References on p . 376.
374
u s u a l l y c o n t a i n e d i n t h e m o b i l e phase a t a c o n s t a n t c o n c e n t r a t i o n , w h i l e t h e c o n t e n t o f t h e o r g a n i c s o l v e n t ( a ~ e t o n i t r i l e l l ~ -methanol ~~~, o r i s o p r o p a n o l 'I6) changed 1 i n e a r l y d u r i n g g r a d i e n t e l u t i o n . Reversed-phase g r a d i e n t e l u t i o n chromatography on o c t y l - and o c t a d e c y l s i l i c a columns i s s u i t a b l e f o r t h e e f f i c i e n t and r a p i d a n a l y t i c a l and p r e p a r a t i v e mapping o f complex p e p t i d e m i x t u r e s and u s u a l l y y i e l d s b e t t e r s e p a r a t i o n s i n a s h o r t e r t i m e t h a n o t h e r l i q u i d chromatography t e c h n i q u e s . However, i t would be d i f f i c u l t t o a c h i e v e complete r e s o l u t i o n of a l l p e p t i d e s i n v e r y complex m i x t u r e s , such as i n t r y p t i c d i g e s t s o f l a r g e r p r o t e i n s , where i t c o u l d be necessary t o re-chromatograph a zone o f p a r t i a l l y r e s o l v e d p e p t i d e s i n a second, independent i s o c r a t i c run. To g i v e a few examples o f a p p l i c a t i o n s o f t h e reversed-phase chromatography
30
o f p e p t i d e s and small p r o t e i n s , we can mention chromatography o f p r o i n s u l i n , p e p t i d e s o f human f o e t a l g l o b i n 6 8 , n e ~ r o p e p t i d e s ~hypothalamic ~, 01 i g o p e p t i d e s 114 , enkephal i n peptides8' y 9 2 y 1 0 8 ,
argot ens in^"^,
parathyroid"
and a d r e n o c o r t i c o -
t r o p i c 9 2'1 l3hormones, pteroyl-oligo-y-L-g1utaminesg5, growth hormones8',
19-S
t h y l o g l o b u l i n 8 5 , o p i o i d peptides88 , o x y t o c i n , vasopressin, samostating6, t u f t s i n calcitonin112, a p o l i p r ~ t e i n s " ~ ,peptides o f r a t casein82y86, t r y p t i c d i g e s t o f
97
o f egg w h i t e l y s ~ z o m e ~o f~ sheep ~, 19-5 t h y r o g l o b u l i n and
haemoglobin8 4 y 1 0 3 y 1 0 4 ,
o f bovine thyrotrophin117. A h i g h - e f f i c i e n c y separation o f f o u r polypeptides i s shown i n F i g . 21.4 119
.
21.7.
AFFINITY CHROMATOGRAPHY OF PROTEINS
A f f i n i t y chromatography i s a h i g h l y s e l e c t i v e s e p a r a t i o n method t h a t a l l o w s t h e p u r i f i c a t i o n o f p r o t e i n s , p r o t e i n - b a s e d enzymes and hormones and o t h e r b i o p o l y m e r s on t h e b a s i s o f t h e i r b i o l o g i c a l f u n c t i o n s o r i n d i v i d u a l chemical s t r u c t u r e s . T h i s t e c h n i q u e makes i t p o s s i b l e t o remove l a r g e amounts o f cont a m i n a t i n g substances f r o m t h e s o l u t e s o f i n t e r e s t i n b i o l o g i c a l m a t e r i a l o r t o s e p a r a t e t h e a c t i v e f o r m f r o m t h e denatured forms o f t h e same substance. Theref o r e , t h e m e r i t s o f a f f i n i t y chromatography f o r b i o l o g i c a l r e s e a r c h a r e c o n s i d e r a b l e , and t h i s method has found wide acceptance s i n c e i t s f i r s t i n t r o d u c t i o n 120 about 15 y e a r s ago
.
I n p r i n c i p l e , t h e method i s based on t h e i n t e r a c t i o n s between a b i o s p e c i f i c l i g a n d c o v a l e n t l y bonded t o a s u i t a b l e s u p p o r t and t h e substance o f i n t e r e s t , t h e s p e c i f i c s t r u c t u r e o f which " f i t s " t h e s t r u c t u r e o f bonded ( i m m o b i l i z e d ) l i g a n d . F o r example, an enzyme bonded as t h e l i g a n d can be used f o r t h e p u r i f i c a t i o n o f a s u b s t r a t e analogue, i n h i b i t o r o r c o f a c t o r , o r an a n t i b o d y bonded as t h e l i g a n d may be s u i t a b l e f o r t h e p u r i f i c a t i o n o f an a n t i g e n o r o f a v i r u s , a n u c l e i c a c i d as t h e l i g a n d f o r p u r i f i c a t i o n o f n u c l e i c a t i d polymerase o r o f a b i n d i n g protein, etc.
375
2
I
0
1
8
I
16
t(min)
I
24
F i g . 21.4. S e p a r a t i o n o f f o u r p o l y p e p t i d e s by reversed-phase g r a d i e n t - e l u t i o n chromatography. Column: M i c r o Pak MCH, 150 x 5.4 mm I . D . G r a d i e n t : l i n e a r , 18-60% B i n 42 min (1% B/min). S o l v e n t s : A, triethylammonium phosphate b u f f e r (pH 2.2); B, a c e t o n i t r i l e . F l o w - r a t e : 1 ml/min. D e t e c t i o n : UV a t 210 nm. Peaks: 1 = bombesin; 2 = s o m a t o s t a t i n ; 3 = i n s u l i n ; 4 = glucagon. (Reproduced f r o m r e f . 119 w i t h p e r mission.) The use o f t h e method c o n s i s t s i n s e v e r a l c o n s e c u t i v e s t a g e s : ( a ) A s u i t a b l e b i o s p e c i f i c l i g a n d i s c o v a l e n t l y bonded t o a chromatographic bed m a t e r i a l ( m a t r i x ) , such as an agarose g e l (Sepharose 4B, Sepharose CL), p r e v i o u s l y a c t i v a t e d f o r e a s i e r c o v a l e n t attachment o f t h e l i g a n d . I t i s i m p o r t a n t t h a t the attached, "immobilized", l i g a n d r e t a i n s i t s s p e c i f i c b i n d i n g a c t i v i t y f o r t h e substance o f i n t e r e s t . ( b ) The g e l w i t h t h e " i m m o b i l i z e d " l i g a n d i s packed i n t o a column o f s u i t a b l e dimensions, t h e sample i s a p p l i e d , f r o m which substances c a p a b l e o f b i o s p e c i f i c i n t e r a c t i o n s w i t h t h e i m m o b i l i z e d l i g a n d become adsorbed on t h e column, w h i l e t h e non-sorbed i m p u r i t i e s a r e washed away f r o m t h e column w i t h a s t a r t i n g b u f f e r . References on p . 576.
376
( c ) E l u t i o n of t h e sorbed substances from t h e column u s i n g e l u e n t s t h a t decrease t h e a f f i n i t y f o r c e s between t h e substances and t h e immobilized l i g a n d . This i s u s u a l l y achieved by e i t h e r a stepwise o r a continuous change i n propert i e s of t h e e l u e n t and s e l e c t i v e o r n o n - s e l e c t i v e e l u e n t s can be employed, depending on t h e n a t u r e o f t h e b i n d i n g between t h e l i g a n d and s o l u t e s . Nons e l e c t i v e e l u t i o n methods i n v o l v e pH o r i o n i c s t r e n g t h g r a d i e n t s . A change ( u s u a l l y a decrease) i n pH d u r i n g g r a d i e n t e l u t i o n a l t e r s t h e degree o f i o n i z a t i o n of charged groups a t t h e b i n d i n g s i t e s and causes desorption. A g r a d i e n t o f i n c r e a s i n g i o n i c s t r e n g t h (e.g.,
o f sodium c h l o r i d e c o n c e n t r a t i o n ) a l s o leads
t o d e s o r p t i o n of sorbed s o l u t e s a t a c o n c e n t r a t i o n o f about 1 m o l / l . Urea and guanidine h y d r o c h l o r i d e a t h i g h c o n c e n t r a t i o n s a r e u s e f u l e l u e n t s , b u t they should be removed from t h e p u r i f i e d p r o t e i n s immediately a f t e r t h e e l u t i o n , otherwise they c o u l d cause d e n a t u r a t i o n o f p r o t e i n s . S e l e c t i v e ( a f f i n i t y ) e l u t i o n makes use o f 'an i n c r e a s i n g c o n c e n t r a t i o n g r a d i e n t o f an e l u t i n g agent t h a t e i t h e r competes f o r b i n d i n g t o t h e adsorbed substance o r f o r binding t o the ligand. To g i v e a few r a t h e r a r b i t r a r i l y chosen examples from t h e w e a l t h o f p u b l i s h e d i n f o r m a t i o n , we can mention chromatography o f phosphofructokinase on immobilized 121 Cibacron B l u e F 36-A u s i n g a l i n e a r g r a d i e n t (0-1.5 m o l / l ) o f ammonium s u l p h a t e , p u r i f i c a t i o n o f d i p e p t i d y l peptidase I V on t h e immobilized p e p t i d e Gly-Pro-NH(CH2)6-NH u s i n g a g r a d i e n t (0-0.2 m o l / l ) o f sodium c h l o r i d e 1 2 2 , f r a c t i o n a t i o n o f p r o t e o l y t i c enzymes t r y p s i n and chymotrypsin on immobilized a m i n o c a p r o y l p r o f l a v i n u s i n g a g r a d i e n t (0-0.08 m o l / l ) o f sodium chloride123, f r a c t i o n a t i o n o f Escker i c k i a coZi B aminoacyl-tRNA synthetases on immobilized hexylamine u s i n g a
g r a d i e n t (0-0.4 m o l / l ) o f potassium chloride124, p u r i f i c a t i o n o f a l k a l i n e phosphatase on immobilized t r i a z i n e dyes u s i n g a g r a d i e n t (0-1 m o l / l ) o f potassium c h l o r i d e 1 2 5 , f r a c t i o n a t i o n o f a complex m i x t u r e o f dehydrogenases on immobilized adenosine 5'-monophosphate u s i n g a pH g r a d i e n t (pH 6-10)Iz6, p u r i f i c a t i o n o f t r y p s i n and chymotrypsin on immobilized low-molecular-weight soybean protease i n h i b i t o r s u s i n g a pH g r a d i e n t (pH 8-4)127 and p u r i f i c a t i o n o f i n d o l ethylamine N-methyltransferase on immobilized S-adenosylhomocysteine128. I n most o f these a p p l i c a t i o n s , agarose g e l s were used as substrates. Comprehensive i n f o r m a t i o n about t h e v a r i o u s techniques and a p p l i c a t i o n s o f 129 a f f i n i t y chromatography can be found i n a monograph by Turkovd
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