key: cord-0039747-y5vus4c8
authors: nan
title: Chapter 12 Compound Biopolymers and Biooligomers
date: 2009-03-18
journal: nan
DOI: 10.1016/s0301-4770(08)61452-9
sha: 30e0999c8c0edc578d19e730dbd8c95c8f2498bc
doc_id: 39747
cord_uid: y5vus4c8

This chapter is devoted to the separation of simple saccharides. In this chapter, the rapid chromatographic separation of natural oligomeric or polymeric compounds containing important molecular moieties of a different type are discussed, such as nucleoprotein complexes, glycolipids, glycopeptides and glycoside oligomeric derivatives. In addition, separations of several natural complex substances that are not well known are discussed. This chapter concludes with a brief discussion on the separation techniques used for the miscellaneous polymeric and oligomeric substances.

, a f f i n i t y chromatography etc.] were a l s o t e s t e d and s u c c e s s f u l l y used. Some o f these methods w i l l be reviewed i n t h e f o l l o w i n g sections. 12 . 1.1 Size exclusion chromatography 12.1.1.1 

The f i r s t experiments were undertaken w i t h p l a n t viruses. An example o f t h e i r r a p i d mutual separation u s i n g CPG and only h y d r o s t a t i c pressure i s i l l u s t r a t e d by Fig. 12 mosaic v i r u s (SBMV, kfr = 6.6 lo6; cf., Edsall, 1953) , tomato bushy s t u n t v i r u s (TBSV, M~ = 8.9 lo6; cf., Edsall, 1953) , tobacco mosaic v i r u s (TMV, kfr = 39. 4 . 10 ; cf., Caspar, 1963) , and t u r n i p y e l l o w mosaic v i r u s (TYMV, Mr = 5.4 lo6; c f . , Matthews, 1970) . (Smith e t al., 1959; R'dsch, 1963) . I t seems probable t h a t (CPG 10-1250, Corning Glass An e l u t i o n p a t t e r n was published by H i a t t e t a l . (1971) mu1 t i p l e hydrogen bonds between the pol yether and the el ectronegative oxygen atoms i n the -Si02-repeating structure of the glass account f o r the adsorption of poly(ethy1ene oxide). By competing for binding positions on the glass surface, poly(ethy1ene oxide) thus effectively blocks the adsorption of rabies virus.

polymers hydrogen bond t o surface silanol groups of CPG, decreasing the charge on the surface and preventing the adsorption of viruses and proteins. However, this i s unfortunately a reversible process and PEG elutes from columns during continuous use. I n s p i t e of t h i s , Darling e t a l . (1977) described the rapid purification of an RNA tumour virus and proteins by IIP-SEC on bead columns made of CPG treated with PEG. Avian myeloblastosis virus (AMV) and hamster melanoma virus (HaMV) were purified from plasma proteins and tissue culture media components. The purified HaMV was s t i l l infectious, and the AMV-associated RNAdirected DNA polymerase ("reversed transcriptase") showed an 1100-fold purification of the virus a f t e r one column treatment. The time required for column purification was 5 min and electron microscopy of the purified virus showed intact particles.

The CPG (80-120 or 120-200 mesh) f o r t h i s treatment was purchased from Analabs (North Haven, CT, U.S. A.) or Electro-Nucleonics (Fairfield, NJ, U.S.A.) .

The undesirable adsorption of CPG was greatly reduced by a modification of the method of Hawk e t a l . (1972) , consisting in pre-treatment w i t h PEG solution.

Pre-treatment procedure (Darling e t al., 19771. A 100-9 amount of CPG beads was added t o 500 ml o f degassed solution of 3% PEG 20M in d i s t i l l e d water. The suspension was maintained under vacuum t o ensure that a i r held in the pores was removed t o allow PEG t o enter. The suspension was shaken occasionally t o f r e e any a i r bubblesadsorbed on the beads. After 30 m i n the suspension was connected t o a vacuum line overnight. The excess of PEG was removed from the beads by repeated washing with d i s t i l l e d water until foam was no longer observed. The beads were a i r dried under vacuum (oven drying had t o be avoided!).

Stainless-steel columns 1, 2 or 3 m long and 1/4 in. 0 activity versus protein concentration, ( 2 ) by a difference in UV absorption (A254 -A280) and (3) by electron microscopy. Routinely, only the UV difference According t o Regnier e t a l . (1977) , apparently polyethylene glycol (PEG) Eluting virions were detected ( 1 ) by assay for detergent-requiring polymerase Choppin, 1974, 1977) . Ac- The pore s i z e s were 10 and 30 nm, r e s p e c t i v e l y . P r o t e i n s c o n t a i n i n g e x t r a c t s were chromatographed a t a f l o w -r a t e o f 1 ml/min w i t h a l i n e a r g r a d i e n t from 10 t o 60% o f s o l v e n t B i n A f o r 24 min. Solvent A was 12 mM HC1 i n t r i p l y d i s t i l l e d water and B was 12 mv HC1 i n ethanol-I-butanol

Sendai v i r u s i s a paramyxovirus, ranging i n s i z e f r o m 150 t o 250 nm ( W e l l i n g B307 (4:1, v/v). Both the organic solvents were obtained from E. Merck (Darmstadt, F.R.G.) . The amount o f proteins t o be analysed was very s m a l l (10-50 pg). The p u r i t y o f the i s o l a t e d p r o t e i n s was checked e l e c t r o p h o r e t i c a l l y using SDS-PAGE. P r o t e i n F2 was i s o l a t e d i n r e l a t i v e l y l a r g e amounts ( t h e y i e l d was about loo%),

whereas the recoveries o f three other p r o t e i n s (Fly HN and M I were r e l a t i v e l y low (from l e s s than 5% t o 50%). The amino a c i d a n a l y s i s o f F2 was presented.

Welling e t a1 . (1983) To remove most o f T r i t o n the tubes were covered w i t h dialysis-membrane tubing and d i a l y s e d overnight a t 4OC against water w i t h Bio-Beads SM 2 (Bio-Rad Labs., Richmond, CA, U. S.A.; cf., Volsky and Loyter, 1978) . The dialysed f r a c t i o n s were reduced and checked by SDS-PAGE.

The detergent e x t r a c t contained F and HN proteins. HN was n o t r e t a i n e d by the column, and e l u t i o n w i t h a s a l t gradient r e s u l t e d i n several peaks, cont a i n i n g mainly o r o n l y F protein. As B r i j 35 detergent d i d n o t absorb a t 280 nm, the same chromatography was performed w i t h 0.1% (v/v) B r i j i n the eluent. A s i m i l a r separation resulted, although some t a i l i n g o f peaks was observed. The ion-exchange procedure described was found t o be a r a p i d method f o r the p u r i f i c a - A stainless-steel column (500 mm x 4 mm I. D.) was slurry-packed with 10-pm Iatrobeads 6RS-8010 porous s i l i c a spheres (Iatron Labs. 1 using tetrabromoethane-2 tetrachloroethylene (60:40) a t 400 kg/cm . The column was washed with isopropanol-hexane (55:45) and equilibrated with the s t a r t i n g solvent (e.g. , isopropanol-hexane-water, 55:44: 1). For elution a 1 inear gradient of isopropanolhexane-water was used (the concentrations of hexane and water were varied, depending on the glycol ipid composition). The gradient was i n i t i a t e d immediately a f t e r injection a t a flow-rate of 2.0 ml/min. Every 0.5 or 1 min one e f f l u e n t fraction was collected in the fraction c o l l e c t o r . After use, the column was regenerated by washing with isopropanol-hexane (55:45) a t a flow-rate of 1 ml/min f o r 60 min and could be used again without any l o s s of efficiency.

bre-coated s i l ica gel G plates, developed with chl oroform-methanol -water GSL with mono-t o dodeca-o r tetrakisdecasaccharides were separated highly reproducibly within 60 min. The method was applied t o preparative separations of highly complex glycolipids with blood group a c t i v i t y .

No on-line sample detection was applied. The f r a c t i o n s were analysed by TLC derivatives chromatography of unsubstituted glycosphingol ipids (GSL). A simple derivatization method was sought t h a t would introduce a s u i t a b l e UV chromophore i n t o the glycolipid and allow the u t i l i z a t i o n of a s e n s i t i v e UV detector. In addition, the modifying group should be easily s p l i t off a f t e r the chromatographic separation. This was believed t o be a practical means of taking advantage of HPLC methods in glycol ipid biochemistry. Evans and McCluer (1972) reported a system f o r the separation of neutral glycolipids by HPLC of t h e i r benzoylated derivatives, prepared by reaction with benzoyl chloride, in a manner similar t o t h a t described by Acher and Kanfer (1972) . They demonstrated t h a t benzoylated cerebrosides could be readily separated i n t o two f r a c t i o n s by HPLC on a s i l i c a gel column. I t was o r i g i n a l l y believed t h a t the derivatives prepared were only 0-benzoyl derivatives and t h a t the parent cerebrosides could be simply regenerated by c a t a l y t i c deacylation with sodium methoxide in methanol. However, McCluer and Evans (1973) found t h a t the reaction of cerebrosides containing non-hydroxy f a t t y acids with benzoyl chloride resulted in acylation of the amide in addition t o the normal 0-acylation (and mild a l k a l i treatment of the N-diacyl derivative resulted in t h e formation of N-benzoylpsychosine). Cerebrosides containing hydroxy f a t t y acids did not

The lack of a s u i t a b l e on-line detection method i s a disadvantage of the B314 create the undesired N-benzoyl derivatives. Derivatization with benzoic anhydride avoided amide acylation even in the presence of non-hydroxy f a t t y acid components; however, t h i s l a s t reaction was n o t sufficiently f a s t . McCluer and Evans (1973) described the benzoylation of cerebrosides and derivatives that contained hydroxy and non-hydroxy f a t t y acids were isolated by HPLC with UV detection.

BenzoyZation procedure. Cerebroside samples (0.1-1 .O mg) were benzoylated with 0.6 ml of benzoyl chloride-pyridine (1:5, v/v) f o r 1 h a t 6OoC (similar amounts were benzoylated with 0.6 ml of 5% benzoic anhydride in pyridine for 18 h a t l l O°C ) . Two procedures were utilized t o purify the benzoylated products, as follows. ( A ) The reaction mixture was dried under a stream of nitrogen and the residue was dissolved in 5 ml of hexane and successively washed with 3 ml each of 95% methanol saturated with Na2C03, 0.6 M HC1 in 95% methanol and 95% methanol. The hexane layer was dried under a stream of nitrogen. ( B ) The reaction product was refluxed under methanol and chloroform was used f o r extraction; a f t e r successive washing of the lower layer, the solution was evaporated and dissolved in hexane.

For HPLC a 50 cm x 2.1 mm I.D. stainless-steel column was dry-packed with Zipax pellicular packing (Instrument Products Division, DuPont, Wilmington, DE, U.S.A.) and eluted with 0.13% methanol in pentane a t a flow-rate of 1.5 ml/min. a bonded polar phase consisting of 3-aminopropylsilane groups) was used f o r the separation of perbenzoylated galactosyl-and glucosylceramides (McC1 uer and Evans, 1976) . Suzuki e t a l . (1976) also described the separation of molecular species of glucosylceramide by HPLC of their benzoyl derivatives.

Jungalwala e t a l . (1977) reported the determination of less than 1 nmol of perbenzoylated cerebrosides by HPLC using gradient elution. Both hydroxy and non-hydroxy fatty-acid-containing cerebrosides were analysed with UV detection a t 230 or 280 nm. The quantitative range of the method was 0.5-10 nmol of cerebrosides. The detection limit f o r injected samples was about 1 pmol and the analysis time was less than 5 min. No preliminary purification of the cerebrosides from other lipids in brain extracts was necessary. The moisture-free cerebrosides were f i r s t benzoylated with 50 ~1 of 10% benzoyl chloride i n pyridine and then separated on a 50 cm x 2.1 mm 1.0. column of 27 pm Zipax porous layer s i l i c a beads. If gradient elution with 2.8-5.5% dioxane in hexane was applied, monitoring a t 230 nm (i.e., a t the absorption maximum of benzoylated cerebrosides) was possible. The column was regenerated by reversing the gradient for 1 min and equilibrating the column w i t h 2.8% dioxane i n hexane f o r 3-4 min. This procedure was a t least 10 times more sensitive than isocratic elution with detection at 280 nm (McCluer and Evans, 1976) . Jungalwala e t a l . (1977) also de-In addition to Zipax, a MicroPak NH2 column (microparticulate s i l i c a gel with scribed a v a r i a n t o f g r a d i e n t e l u t i o n w i t h d e t e c t i o n a t 280 nm, i n which a l i n e a r g r a d i e n t o f 2-7% aqueous e t h y l a c e t a t e i n hexane was used f o r e l u t i o n .

The column was regenerated t o i t s o r i g i n a l p o l a r i t y by r e v e r s i n g t h e g r a d i e n t f o r 1 min and e q u i l i b r a t i n g the column w i t h 2% aqueous e t h y l a c e t a t e i n hexane f o r 3-4 min. The methods described a r e a p p l i c a b l e t o b i o l o g i c a l m a t e r i a l s cont a i n i n g minute amounts o f cerebrosides.

perbenzoylated n e u t r a l GSL were published by Ulman and McCluer (1977, 1978) ; the f i r s t paper describes HPLC w i t h monitoring a t 280 nm and the second a t 230 nm. An example o f separations reported i n the f i r s t paper i s i l l u s t r a t e d i n S i m i l a r improved methods f o r the q u a n t i t a t i v e a n a l y s i s o r microanalysis o f

The Per-0-benzoyZation procedure (Gross and MeCZuer, 1980) . Conversion of perbenzoylated products t o the parent GSL (Gross and MeCZuer, 1980 Preparative and a n a l y t i c a l HPLC o f modified GSL using a s i m i l a r method (per-0-

McCluer and Ulman (1980). i s o c r a t i c a l l y w i t h 5% isopropanol i n hexane-dichloroethane (2:l). Before the next analysis, the column was r e a c t i v a t e d w i t h t h e i n i t i a l s o l v e n t f o r 15 min. 

A very rapid method f o r the separation of gangliosides by anion-exchange chromatography on strongly basic Mono Q resin (9.8 pm, volume of the pre-packed bed = 1 ml) in combination with FPLC equipment (Pharmacia, Uppsala, Sweden) was presented by Mdnsson e t a l . (1985) . The gangliosides were separated i n t o mono-, di-, t r i -and tetrasialoganglioside f r a c t i o n s by a discontinuous gradient of potassium a c e t a t e in methanol. The separation was completed i n a volume o f 50 ml (25 min). The Mono Q column was washed before use with 10 ml of methanol, 50 ml of 1 M potassium a c e t a t e in methanol and 10 ml of methanol a t a flow-rate of 1 ml/min. A ganglioside sample dissolved in chloroform-methanol (1:2, v/v) was introduced on t o the column a t a flow-rate of 1 ml/min. The gangliosides were eluted with a stepwise gradient from 0 t o 225 m~ potassium a c e t a t e i n methanol a t a flow-rate of 2 ml/min. Fractions o f 1 ml were collected and aliquots were assayed by HPTLC with I-propanol-0.25% aqueous potassium chloride (3: 1 , v/v). The pooled f r a c t i o n s were dialysed against water and f u r t h e r assayed. After each analysis the column was washed w i t h 5 ml of 1 M potassium a c e t a t e in methanol and 20 ml of methanol. Very good and rapid separations of gang1 iosides were achieved on Mono Q, b u t the application of t h i s strongly basic anion exchanger had some disadvantages: p a r t of the tetrasialoganglioside GQIb was eluted among t h e monosialogangl iosides, probably owing t o lactonization of some of the s i a l i c acid residues, and a c e r t a i n non-specific adsorption e f f e c t a l s o appeared.

the separation o f gangliosides. Linear gradient elution using ammonium a c e t a t e in methanol resulted i n the complete separation of mono-t o pentasialogangliosides in 35 min. The separation obtained, i l l u s t r a t e d in Fig. 12 .3, did not suffer from the undesirable c a t a l y t i c a c t i v i t y of the anion exchanger on the s e n s i t i v e gangliosides. Fractions were assayed by TLC and the technique of "ganglioside mapping" was employed (Ivamori and Nagai , 1978a) f o r t h e i r detailed identification. The rapid preparative separation of gangliosides on medium-basic anion exchangers was described by h i d e t a l . (1986b).

h i d e t a1 . (1986a) applied the medium-basic anion exchanger DEAE-Spheron t o

Ion-exchange chromatography permits only t h e group separation of gangliosides according t o t h e number of s i a l i c acid residues, and these groups usually contain more than one ganglioside species. Unfortunately, the present methods of IEC are not able t o f r a c t i o n a t e these groups i n t o pure individual gangliosides and a subsequent separation i s necessary. Some chromatographic methods allowing the d i r e c t fractionation of gangliosides (without any pre-fractionation by IEC) have been investigated. However, t h e combination of IEC w i t h LSC ( s i l i c a gel chromatography) was used most often and became almost customary. For detection a moving v i r e was applied with a flame ionization detector. In addition t o three unknown peaks, s i x known gangliosides were identified. In a separate chromatography of neutral glycosphingolipids, one unknown peak appeared in addition t o four known species, when neutral chloroform-methanol ( 3 : l ) was used f o r elution. Also here the same universal detector was employed f o r monitoring. Tjaden e t a l . (1977) recommended the method f o r both analytical and micropreparative purposes. Watanabe and Tomono (1984) developed a "one-step" fractionation of neutral and acidic glycosphingolipids by HPLC. They connected one column of DEAEderivatized controlled-pore glass (CPG) s e r i a l l y with two columns of underivatized CPG. A mixture of gangliosides and neutral glycosphingolipids was loaded on the DEAE-CPG column and washed using gradient elution based on chloroformmethanol -water with increasing methanol and water contents , f o l l owed by a second gradient with an increasing amount of lithium a c e t a t e solution. In the f i r s t gradient elution neutral glycol ipids (mono-t o hexaglycosylceramides) were separated within 80 min, and in the second mono-t o tetrasialogangliosides were separated within 60 min. . The e f f l u e n t was collected in a fraction c o l l e c t o r and each fraction (or every second f r a c t i o n ) was examined by TLC o r using resorcinol reagent (Suzuki , 1964) .

Controlled-pore glass (CPG-10) of pore diameter 75 A, 200-400 mesh, and A very good separation was obtained using t h i s one-step f r a c t i o n a t i o n method.

and n e u t r a l and a c i d i c g l y c o l i p i d s i s o l a t e d from r a t kidney. The procedure was found t o be h i g h l y reproducible. 12.2.3.3 Individual gangliosides were collected and identified by TLC and GC of their monosaccharides. Quantitative results were obtained in this method, together with a mrked increase in sensitivity in comparison with conventional analytical methods.

Nakabayashi e t a l . (1984) studied the analysis and quantitation of gangl iosides as p-bromophenacyl derivatives by HPLC. This method was found t o be highly sensitive, and involves a simple and specific derivatization of the carboxylic group. The molar absorptivity of the derivatives a t maximum absorption (261 nm) was about 23 000. The acidic fractions from IEC pre-fractionation on DEAE-Sephadex could be directly derivatized and the reaction mixtures directly injected into the HPLC equipment without any preliminary purification. Both normal-and reversed-phase modes could be applied for the separation of gangl ioside mixtures.

For HPLC a 30 cm x 2 mm I.D. reversed-phase 10-um VBondapak c18 column (Waters o f drop1 e t counter-current chromatography (DCC) f o r the separation of acidic glycosphingol ipids from brain and neutral glycosphingol ipids from mammalian erythrocytes (blood group substances), Pick e t a l . (1984a,b) used a method for overpressured TLC (Tyihak e t a l . , Otsuka and Yamakawa (1981) and Otsuka e t a l . (1983) described the application 12.2.5 AppZications of HPLC of gZycosphingoZipids 12.2.5.1 Discoveries of noveZ glycosphingolipids progressive process: nearly every year brings knew knowledge i n t h i s f i e l d and new species are discovered and structurally characterized; many o f them are gangl i osi des. Ando e t a l . (1976b) described the existence and structure of glucosamyl-1 actosylceramide acto-N-triose( 1I)ceramide , amino CTH-q in human erythrocyte membranes as a possible precursor of paragloboside and group-active glycol ipids; high-speed Iatrobeads chromatography was used f o r i t s purification. Ando and Yu (1977) reported the isolation and structural characterization of a t r i s i a l oganglioside, GTla, from human brain; i t represented 0.6% of the total gangliosides. Also here Iatrobeads chromatography was applied as a final fractionation and purification step of the trisialogangl ioside fraction obtained by IEC. Ivamori and Nagai (1978b) presented results o f the isolation and characterization of a gangl ioside (monosialosylpentahexaosylceramide) from human brain. Using a combination of IEC and Iatrobeads chromatography, approximately 2.1 umol of an unknown ganglioside were obtained from 1 kg of human brain (this amount comprised 0.09% of the total lipid-bound s i a l i c acid in the brain). The characterization procedures were described. Ivamori and Nagai (1981~) studied monosialogangl iosides of rabbit skeletal muscle and characterized a novel N-acetyl neuraminosyl lacto-N-noroctaosyl ceramide. After IEC (DEAE-Sephadex, DEAE-Sepharose) the monosial ogangl ioside fraction was further fractionated by LCS on Silica gel 60 and Iatrobeads columns.

As in the above-mentioned papers, also here the purity of the obtained fractions was examined by TLC. The newly isolated ganglioside represented 5.1% of the monosialogangl ioside fraction. Chien and Hogan (1983) described a novel pentasia-The investigation of glycosphingol ipids i s a very systematic, thorough and 

The a n a l y t i c a l chromatographic separation o f glycosphingol i p i d s has been Svennerholm (1963 Svennerholm ( , 1964 , and t h e IUPAC- Commissions (1977 Commissions ( , 1978 recommended a u n i f i e d nomenclature f o r 1 i p i d s .

The Gram-positive and Gram-negative bacterial cell walls have a common feature, viz. , the rigid structural framework consisting of parallel polysaccharide chains covalently cross-linked by peptide chains, and this framework constitutes 10-50% or even more of the weight of the cell wall. W e shall n o t comment here on the accessory components, which are different i n the two types of bacterial c e l l , b u t will focus our attention on the main macromolecular framework, a heteropolymer, which i s called peptidoglycan or murein (murus means wall in Latin). The peptidoglycan forms a completely continuous covalent structure around the cell. For example, Gram-positive bacteria are encased in up to 20 layers of cross-1 inked peptidoglycan (Lehninger, 1978b) . The basic recurring unit in peptidoglycans (creating long polysaccharide chains) i s a disaccharide of N-acetyl-D-glucosamine and N-acetylmuramic acid in a B( 1 4 ) 1 inkage, t o the carboxyl group of which a peptide i s attached by means o f the N-terminus. The peptidoglycan cross-1 i n k i n g in the StaphyZococcus aureus cell wall i s given below as an example (according t o Strominger and Ghuysen, 1967 ; see also peptidoglycan biosynthesis, dealt with by Strominger e t a l . 1967). Rogers and Perkins ( Leive (1973) and ]. (Kotani e t al., 1983; Adam and Lederer, 1984; Lederer, 1986; Jeiek, 1986) MaSek e t Adam and Lederer, 1984; Lederer, 1986) . Ovchinnikov , L e f r a n c i e r and Lederer, 1981; Straka 1983; JeSek, 1986 ).

a c t i v i t i e s i n t h e immune response process kf., e.g., the review (460 references) by Leclers and Vogel (1986) ]. S i m i l a r a c t i v i t y was found, e.g. , i n t h e n a t u r a l t e t r a p e p t i d e t u f t s i n (Thr-Lys-Pro-Arg) , which i s a phagocytosis-stimulating , 1982) were prepared and tested in various forms, and a l s o in the form of virosome influenza vaccine. Masihi and Lange (1986) reported the stimulation of non-specific resistance against respiratory infections by immunomodulators. Eleven MDP analogues were found t o be e f f e c t i v e (when combined with 6,6'-trehalose dimycolate) against influenza virus o r @cobacteriwn tuberculosis infection.

JeZek e t a l . (1986, 1987) 

proposed for searching f o r determinants. Jemmerson and Paterson (1986b) presented a method for mapping epitopes on a protein antigen by partial proteolysis of the antigen-antibody complexes, in which chromatography played an important role. An antibody bound t o a protein antigen showed a s t e r i c hindrance effect and decreased the rate of proteolytic cleavage of the antigen in regions involved i n the antibody-antigen contact. RP-HPLC of the partial digest has made i t possible to identify the contact area according to the relative amount (peak heights) and composition of the isolated peptides of the antigen, provided that the primary structure of the antigen was known and that a protease digest of the single antibody and the single antigen were chromatographed in preliminary experiments in order to localize their peptide peaks in the chromatogram. Cytochrome c and monoclonal antibody against i t were used in these experiments.

A 30-min tryptic digestion was found t o be optimal. For HPLC a 250 mm x 4.6 mm I.D. column of C I 8 Spherisorb ODs-1 (Custom LC, Houston, TX, U.S. A.) was eluted with a linear gradient from 0 t o 70% acetonitrile (0.1% TFA) during 90 min a t a flow-rate of 1 ml/min. UV detection a t 214 nm was applied. Collected peaks of the antigen were hydrolysed and analysed i n an amino acid analyser. The applicability of t h i s method was discussed in detail. Another, b u t very expensive and time-consuming, experimental method f o r determining the contact area between the antibody and antigen i s X-ray crystallography of the antibody-antigen complex (Amit e t al., 1985) , from which the epitopes could be derived. This method i s very precise b u t i t cannot be generally used.

The theoretically proposed or practically found antigen determinants are the starting p o i n t f o r the peptide synthesis (Shinnick e t a l . , 1983; Walter, 1986) .

However, so f a r the purified peptides corresponding t o protective epitopes often possess poor immunogenicity, " a t l e a s t partly because of the removal of components carrying adjuvant activity of the original vaccines (Kotani e t a l . , 1986) ; hence the development of new types of vaccines requires studies of chemically well defined i mmunoadj uvants , which ef f ec t i vel y potentiate the immunogenic i ty of protective epitopes". Also, the effect of additivity or synergism caused by a large number of determinants on the same natural macromolecular antigen contributes t o the stronger antigenicity of natural antigens i n comparison with single synthetic pepti des.

The chromatography of synthetically prepared peptides f o r vaccination does not d i f f e r from the separation techniques for any other simple peptides, which were described in detail in Chapter 11, and therefore in the following part of this section only a few examples of synthetic vaccines will be presented. 12.3.4.2 Examples There a r e two surface g l y c o p r o t e i n s i n i n f l u e n z a v i r u s , haemagglutinin and neuramidinase. The f i r s t p r o t e i n i s the major v i r a l surface a n t i g e n and w i l l be d e a l t w i t h l a t e r (cf., M i l l e r e t Shapira e t al., 1984; Wabuke-Bunoti, 1984a,b; HamSikovd e t al., 1986 HamSikovd e t al., , 1987 . Pro and t h r e e T y r residues ( i m p o r t a n t f o r a n t i g e n i c i t y ) and a f o l d e r corner i n t h e X-ray s t r u c t u r e ; t h i s was suggesting because t h e r e was a chance t h a t t h i s Arnon and Shapira (1984) d e a l t w i t h a n t i -i n f l u e n z a s y n t h e t i c vaccines and reported t h a t a t l e a s t twelve (300 in 0-40 min a t a flow-rate of 1 ml/min; solutions: A = 0.08% TFA in watern-propanol (9: 1 ) ; B = 0.05% TFA in water -n-propanol (1:l). The TyrI6' peptide was linked with human serum albumin using bis-diazotized benzidine. In preliminary experiments, the peptide showed good reactivity with an antiserum directed toward the complete virus, b u t immunization with the albumin-conjugate (peptide t o protein ratio = 8: 1 ) gave no virus neutralizing anti bodies. Krchfidk and Ma19 (1986) used modern technology t o prepare and t e s t synthetic antigen fragments i n which the isolation of the real intact-protein antigen was not necessary. The principle of t h i s approach can be illustrated by the following steps: ( 1 ) determination of the DNA sequence expressing the antigen; (2) selection of the corresponding protein fragment(s1 based on theoretical translation; Ddlling e t a l . (1986) described the synthesis of peptide sequences belonging column was used ( c f . , Fig. 12 .61, eluted with a gradient of Z%/min of B (Boyle e t al., 1983; Klempnauer e t al., 1983; MalJ and KrchRdk, 1984) . Straka (1983) t h e p r e p a r a t i o n o f fragments o f b a c t e r i a l c e l l w a l l peptidoglycan and analogues ( i n Czech; 280 references) , Adam and Lederer (1984) (for the parent FA n= 1 and f o r Pt-oligoglutamates n= 2-9), and have important biological functions as coenzymes essential in the synthesis of proteins and nucleic acids (Baugh and Drumendieck, 1971 ) and other functions. Because the FA derivatives are acids or 01 igoacids, IEC on microparticulate bonded phases was used for their rapid separation f i r s t (Reed and Archer, 1976 ; Stout e t a l . , 1976). Naturally, the retention of PtGlun in anion-exchange chromatography depends on the number of carboxyl groups, being highest with species containing most Glu residues.

Bush e t a l . (1979) studied the retention behaviour of PtGlu, in RPC on an ODS-silica column and found t h i s method t o be very suitable f o r good resolution and rapid analysis. When the carboxylic groups are largely undissociated ( a t pH 2 ) , the retention of PtGlun increases with the number of Glu residues and the elution order parallels that in IEC. A t sufficiently high pH (e.g., a t pH 4.5) the carboxilic groups are dissociated and the elution order i s reversed (cf., Fig. 12.7) . The logarithm of the capacity factor i s linearly dependent (with the exception o f FA) on the number o f Glu residues over a wide range of eluent pH ( c f . , Fig. 4 in the original paper). As can be seen from Fig. 12 .8, a t pH 6 ( a t which the carboxyl groups are almost completely dissociated) the retention decreases with increasing number of Glu residues, so t h a t both methods (IEC and RPC) complement each other. In general, the efficiency of IEC with bonded phases appears t o be higher than that o f RPC a t low pH on ODS with the same particle size and column dimensions. IEC i s recommended when the elution by a Braton-Marshall procedure s l i g h t l y modified by Eto and Krumdieck (1980) and were p u r i f i e d by Bio-Gel P-2 polyacrylamide gel chromatography. A l l t h e a n a l y s e s were performed on a 25 cm x 0.46 cm I.D. column packed w i t h 

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