hydrophobic (interaction) chromatography c).hydrophobic (interaction) chromatography c). j.-l....

BIOCHIMIE, 1978, 60, 1-1_5. Revue

Hydrophobic (interaction) chromatography c*). J.-L. OCHOA * * l n s t i t n l o d e Q u i m i c a .

U n i v e r s i d a d N a c i o n a l A u t o n o m a de M e x i c o (UNAM) , C i u d a d U n i v e r s i t a r i a , M e x i c o , D.F.

I n t r o d u c t i o n .

The i so la t ion and pur i f ica t ion of mac romole - cules by b i o c h e m i c a l f r ac t iona t ion techniques , l ike ion-exchange c h r o m a t o g r a p h y , gel f i l t ra t ion (molecular-s ieve c h r o m a t o g r a p h y ) , aff ini ty chro- Ina tography, e lec t rophores i s , etc., is p r i m a r i l y dependen t on the i r b io logica l and phys i cochcmi - cal p rope r t i e s [1-5].

Based on biospeci f ic in t e rac t ions [6], aff ini ty c h r o m a t o g r a p h y has been cons ide red as one of the most effective sepa ra t ing methods. However , ser ious d i sadvan tages are found upon its app l ica - t ion, due ma in ly to undes i r ab le non-biospeci f ic adso rp t ion [7] a t t r ibu ted to the cha rac te r i s t i c s of the ma t r i x ov suppor t , and the na ture of the l igand and space r - a rm in t roduced to b~idge the l igand f rom the ma t r i x backbone . Moreover, once a p ro t e in is a t t ached to an immobi l i zed l igand, for w h i c h it sho~vs affinity, the p rope r t i e s of the suppor t may change and become l ike those of an ion-exchanger , i n t e r f e r ing w i th the chromatogra - ph ic process .

The bel ief tha t such in te r fe rences could be adequate ly con t ro l l ed by e l imina t ing ionic groups in the ma t r ix a n d / o r in the spacer -a rm, brought as a consequence other types of undes i r ab le effects closely re la ted to the ex ten t of h y d r o p h o - b i c i ty of the space r - a rm employed [57]. Yon [93, and Er-eI et aL [10], r epo r t ed that by coupl ing dif ferent t ypes of space r -a rms (vary ing in hyd ro - phob ic i ty ) to an ine r t mat r ix , potent adsorbents for p ro te ins we re obta ined. The adsorp t ion mechan i sm seems to be based, fundamenta l ly , on h y d r o p h o b i c in te rac t ions be tween the p ro t e in and the adsorben t , and it has been demons t r a t ed that it can be pos i t ive ly exp lo i ted for sepa ra t ion p u r - poses [11, 105]. I n this way , a novel t echn ique w h i c h takes advantage of the h y d r o p h o b i c i t y of

* Th i s ar t ic le is a c o m p l e m e n t a r y par t to m y con t r i - b u t i o n at the F o r u m des Jeunes , in L y o n , France (July 6-8, •977).

** Present address : Facultd de Mddecine de Stras- bourg, I n s t i t u t de Clfimie Biologique, 11 rue Humann, 67000 Strasbourg, France.

biomolecules , has been i n t roduc e d as a comple- ment to the other, r ou t ine ly employed , sepa ra t ing methods men t ioned above.

Since the sepa ra t ion is based on h y d r o p h o b i c in te rac t ions , th is t echn ique rece ives a number of names 'which in tend to desc r ibe the p r i n c i p l e and the p a r a m e t e r s involved in the sepa ra t ion process , though all of them are en t i re ly or pa r t ly dea l ing wi th the concep t of h y d r o p h o b i c i t y :

H y d r o p h o b i c c h r o m a t o g r a p h y [11] ; H y d r o p h o - b ic ( in te rac t ion) c h r o m a t o g r a p h y [51] ; Hydro - phob ic sal t ing-out c h r o m a t o g r a p h y [93] ; Phos- pha t e - i nduced p ro t e in c h r o m a t o g r a p h y [54] ; Re- pu ls ion con t ro l l ed c h r o m a t o g r a p h y [94] ; Deter- g e n t p ro t e in - in t e r ac t ion [95, 961 ; H y d r o p h o b i c aff ini ty c h r o m a t o g r a p h y [128] ; etc.

It has been shown [12] tha t a large pa r t of the non-po la r res idues of the amino ac ids in p ro t e ins are exposed to ~vater in ter face , as opposed to the expec ted p re fe ren t i a l loca t ion of the h y d r o p h o b i c amino ac ids in the i n t e r io r of the b iomolecule . These non-po la r amino ac ids are found in the p ro te in surface fo rming << patches>) of d i s t inc t h y d r o p h o b i c cha rac t e r w h i c h Can account for the b iospeci f ic conformat ion of the p ro te in [13] as we l l as for i ts ab i l i ty to complex or aggregate to o ther types of molecules for ins tance, l ip ids . Pre- sumably , these h y d r o p h o b i c (< pa tches >> are ran- domly d i s t r i bu t ed on the sur face of the b iomolecule. The i r number (possible number of in ter - ac t ing sites) and the extent of the i r h y d r o p h o b i - c i ty ( type and d i s t r ibu t ion of the non-po la r amino acids) should be a cha rac t e r i s t i c of each macro- molecule . Therefore , the i r specif ic s epa ra t ion shou ld be poss ib le wi th an adequate h y d r o p h o - b ica l ly coated suppo r t or mat r ix .

I . T H E BIOLOGICAL ROLE OF THE HYDROPHOBICITY

IN BIOMOLECULES.

There is no doubt that the h y d r o p h o b i e in ter - ac t ions p l ay an i m p o r t a n t role in b io log ica l systems. The membranes in the l iv ing cell are made

1

2 J.-L. Ochoa.

up of ma in ly h y d r o p h o b i c a l l y in t e rac t ing l ip id - l ip ids and l ip id -p ro te ins . The sub-uni ts of large p ro t e in s a re often he ld toge ther by h y d r o p h o b i c bonds , and i t is wel l accep ted that they are im- po r t an t in suppo r t i ng the t e r t i a ry p ro t e in s t ructure [13].

Func t iona l ly , h y d r o p h o b i c in te rac t ions seem to be involved in r ecogn i t ion processes , as for exam- p le in the case of some enzyme-subs t ra te com- p lexes and an t igen-an t ibod ies associa t ions , etc. [14-15, 27-30]. Excep t for the case of membranes , in w h i c h an exclus ive s t ruc tu ra l funct ion has been a t t r ibu ted [31], the re is not much w o r k in co r re l a t ing the h y d r o p h o b i c p rope r t i e s of the b iomolecu les wi th the i r funct ions [32, 99-100]. Therefore , it could be in te res t ing to f ind out w h e t h e r these h y d r o p h o b i c regions in the biomolecules are re la ted to the i r i n t r in s i c b io logica l p roper t i e s , a n d / o r to the i r loca t ion in the cell.

P e r h a p s one of the reasons w h y fe"w sys temat ic s tudies of h y d r o p h o b i c effects have been made, is tha t the h y d r o p h o b i c compounds possess lo"w so lub i l i ty in wa te r . If they are made soluble by the i n t roduc t ion of po l a r groups, they tend to fo rm mice l les as exempl i f ied by the case of soaps. Al though molecu la r d i spe r s ion may be ob ta ined th rough the add i t i on of p o l a r i t y r educ ing agents to the medium, such agents w o u l d also reduce the in t e rac t ion of the h y d r o p h o b i c compounds w i t h p ro t e ins and even a l ter the p ro te in s t ruc ture , w h i c h genera l ly depends on the in tegr i ty of the h y d r o p h o b i c core [13].

A means to obta in molecu la r d i spe r s ion of a h y d r o p h o b i c l igand in aqueous mil ieu, w i thou t the add i t i on of p o l a r i t y r educ ing agents, is to a t tach the l igand to a h y d r o p h i l i c but insoluble p o l y m e r such as agarose. Consequent ly , h y d r o p h o b i c suppor t s can be used not only for sepa ra t ion pur - poses, but also to s tudy the h y d r o p h o b i c i t y of the b iomolecu les and the i r modes of in te rac t ion . In turn, th is may lead to a c lea re r u n d e r s t a n d i n g of the i r funct ions .

II . THE PHYSICOCHEMICAL CONCEPT OF HYDROPHOBICITY.

P e r r i n [16] `was the first to ut i l ize the te rms << h y d r o p h i l i c >> and ¢ h y d r o p h o b i c >> when study ing col loids . He cons ide red them h y d r o p h i l i c if t he i r s t ab i l i ty was re la t ive ly insens i t ive to the add i t i on of e lec t ro lytes , or h y d r o p h o b i c if they exh ib i t ex t reme sens i t iv i ty to a d d e d e lec t rolytes . Later , Langmui r [17], in a c lass ica l expe r imen t , d i scussed p o l a r molecules (fatty acids) in terms of

h y d r o p h i l i c and h y d r o p h o b i c groups. Since then, these te rms have been in every day use.

In general , "when an apo la r group (or h y d r o p h o - bic) is inser ted in to `water, severa l effects can be observed (for r ev iew see ref. 18) :

- - A negat ive un i t a ry en t ropy change ( - - A S ) , `which impl i e s an overa l l inc rease in the degree of order . The en t ropy d i s t r ibu t ion becomes rela- t ive ly i m p o r t a n t as the mo lecu la r weight of the apo la r group increases .

- - A smal l en tha lpy change, usual ly negat ive ( - - A H ) , w h i c h reflects an energe t ic componen t in the in te rac t ion be tween 'water and an apo la r group and opposes the negat ive un i t a ry effect favour ing mix ing of the h y d r o c a r b o n in water .

- - An inc rease in hea t c apac i ty ( + ACp), w h i c h impl ies e i ther an inc reased degree of f r eedom of the v ib ra t iona l and ro ta t iona l mot ion w i t h i n the exis t ing s t ruc ture , or a p rogress ive a l t e ra t ion of an exis t ing s t ruc ture as the t empe ra tu r e is ra ised.

- - A decrease in volume ( - - A V ) , w h i c h indi - cates that the a p o l a r groups and the wa te r molecules have packed together w i t h a con t rac t ion of some s t ruc ture , or that the apo la r groups are accomoda ted into open spaces or voids preex is - t ing `within the w a t e r s t ruc ture itself. The fo rmer is un l ike ly in view of the smal l en tha lpy change involved.

- - F i n a l l y , the p resence of apo la r groups in w a t e r resul ts in an inc rease in the number or s t rength of h y d r o g e n bonds w i t h i n the w a t e r molecules , as has been demons t r a t ed by sp in lat- t ice r e l axa t ion s tudies and fu r the r s u p p o r t e d by Raman measurements [60]. I ts p robab le reason is tha t the h y d r o c a r b o n cha ins res t r ic t the mob i l i t y of w a t e r molecu les in such a `way tha t the covalent cha rac t e r of the h y d r o g e n bound is i nc reased [92].

On the o ther hand , the molecu la r p i c tu re of the h y d r o p h o b i c in t e rac t ion is the reverse of that ob- t a ined w h e n i n t r o d u c i n g apo la r groups into w a t e r (see mechan i sm below). The w i t h d r a w a l of the apo l a r groups f rom the aqueous phase removes r e s t r i c t i ons on h y d r o g e n - b o n d be nd ing and thus, achieves a pos i t ive un i t a ry en t ropy . This suppor t s the observa t ion tha t the h y d r o p h o b i c in t e rac t ion is a spon taneous process . This a ssumpt ion was subs tan t i a l ly demons t r a t ed by F r a n k and Evans in 1945 [20]. Some yea r s la ter , it has been shown [19] tha t the free energy values of the t rans fe r of a l ipha t i c h y d r o c a r b o n s f rom an apo la r med ium to a po l a r one, l ike wa te r , inc reases l i nea r ly wi th inc reas ing number s of (CH 2) m e t h y l e n e groups, and thus the h y d r o p h o b i c i t y of the molecule .

BIOCHIMIE, 1978, 60, n ° 1.

Hydrophobic (interaction) chromatography. 3

III. D E T E R MI NATION OF THE HYDROPHOBICITY OF

BIOMOLECULES.

It is wel l kno~vn that most prote ins conta in a relat ively high propor t ion of amino acids ~dth non-polar chains (table I). Tanford [21], and

TABLE I.

Non-polar (hydrophobic) amino acids commonly found in proteins.

AI anine CH 3 -R

Leucine CH3x / CH-CH2-R

CH 3

Isoleucine CH3-CH2-CH-R

I CH 3

Valine CH3 x CH-R

CH3 /

Proline

Phenylalanine

Tryptophane

CH CH2\ CH_CO0tl CH , /

2XNH

d CH:CH\ CH ,,,CH-CH _ -R "~ CH-CH >" z

/1 CH-CH \\ C -- CH-CH ~-R

CH ~ Ixrfl "~CI-I " % CH=C

Methionine CH3-S-CH2-CH2-R

R -CH-CO0- !

+NH 3

Nozaki and Tanford [22], formulated an experi- mental procedure based more or less on the Kauz- m a n n [13] concept ion wh ich permits the estima- t ion of the amino acid hydrophobic i ty . The method consisted, essentially, in de te rmin ing the solubil i t ies of the amino acids in water as ~vell as in progressively increas ing concent ra t ion of some organic solvents, such as ethanol in water. The solubili t ies of the amino acids were extra- polated to pure organic solvents and then the free

energy value of the t ransfer for the amino acid from pure organic solvent to ~vater was calculated. Using as a reference glycine, and substrac- ring its free energy t ransfer value from that of all the other amino acids, it was possible to formu- late a hydrophob ic i ty scale for amino acid residues ~vhere their free energy t ransfer values become more and more posit ive as the hydrophobic character of the compounds increases [18].

Other at tempts in scal ing the amino acid hydrophobic i ty can be i l lustrated by the ~vorks of Bull and Breese [23] and Bigelov¢ [24]. The former studied the effect of the amino acid on the surface tens ion of water and the latter calculated the average hydrophob ic i ty of several pro te ins by their amino acid composi t ion according to data of Tanford and of Bull and Breese.

A different approach ~vas done in terms of the f requency of non-polar side chains in prote ins [2~], in w h i c h yeas found a var ia t ion from 0.21 to 0.47. Separately, F isher [26] employed the ratio of the volume of the polar groups to those of the non-polar as a hydrophob ic i ty degree, but this idea has the inconvenience , like the one men- t ioned above [25], to consider that a group is ei ther polar or non-polar , ~without any gradat ion between these t ~ o extremes.

Recently, a method for s tudying the magni tude of the in terac t ion bet~veen prote in and a l iphat ic hydroca rbon chains, and thus ind i rec t ly the hydrophobic i ty , ~vas reported [~0]. It is based on the par t i t ion of prote ins in an aqueous two phase system con ta in ing dextran and polyethylene glycol and different : fa t ty esters of polyethylene glycol. However, the measurements depend largely on a cr i t ical chain length which , by this technique, should be greater than 8 carbons.

F ina l ly , an approach to localize the hydrophobic sites on the surface of the prote ins by means of in te rac t ing wi th small molecules, has been at tempted using fluorescent probes [27]. It is not difficult to speculate that ~vith this set of infor- mation, a better comprehens ion of many biological phenomena is near. We do not know yet how the complex enzymatic systems are organi- zed, nor how the recogni t ion bet~veen prote ins occurs to consti tute such enzymat ic complexes after pro te in synthesis. Neither do we have a good explanat ion for the t ranspor t of many substances, i nc lud ing proteins , through the hydrophob ic core of the membrane . And fur thermore, it is possible to believe that hydrophob ic ((patches >> in the membrane (either out or inside) are needed to make possible many of the most common biolo-

BIOCHIMIE, 1978, 60, n ° 1.

4 J . -L . O c h o a .

gical phenomena like the recogni t ion of non-polar substances by membrane receptors and even cell- cell in teract ions.

I V . T H E M E C H A N I S M O F T H E H Y D R O P H O B I C I N T E R -

A C T I O N .

The hydrophob ic in te rac t ion is the result of the adherence of two non-polar groups. The case of detergents can be considered as an example where negative entha lpy changes are observed dur ing the micelle format ion in aqueous solvents [33!. If the adsorpt ion of prote ins to hydrophob ic matrices is considered to be a process of l imited micelle formation, a negative change in the entha lpy value ~vould not preclude the hydrophob ic nature of the b ind ing . In addit ion, this change must be negligible as compared to the value of increas ing ent ropy of the system I20]. Fur thermore , before the in terac t ion the water molecules are forced to keep in orde~ a round the hydrophob ic entit ies (fig. 1) as compared to the order in the bulk. When the hydrophob ic sticks come in contact wi th each

o o o o o o o o o o o

o o o

oeOOooeeoeooo o ~ o • •

o : : ~ i i . . . . . . . . o j _ o o o o o o o o o o o o o o o •

o o o o o o o o o o o O O 0 0 0 0 Q e O 0 0 0 0 0 0 O • 0

D •

a) b) F[~. 1. - - The mechanism of hydrophobic inter-

action. The water molecules around the hydrophobie << yard-sticks >> are doted, for simplicity, in order to distinguish them from those in the bul~. Notice. that the difference bet'ween a) and b) is only the improved degree of disorder (-I-AS) of vcater molecules.

change in entropy. In these condi t ions the reac- t ion proceeds spontaneous ly [51]. In other words, t h e input of energy or chemical work is not neces- sary to make possible the in terac t ion between two hydrophob ic molecules in aqueous solutions.

The contact between two different molecules, like the sub-uni ts in the case of some proteins, is largely dependent on the surface areas occupied by the residues which par t ic ipate in the interaction. The concept of accessible surface area [80j describes the extent to ~vhich prote in atoms can form contacts wi th water , and is related to hydrophobic free energies [81]. In any case, the asso- ciat ion of pro te in sub-units , whe ther by van der Waals contacts, electrostatic forces a n d / o r hydrophobic in teract ions , leads to a reduct ion of the surface area accessible to the solvent ~vhen the two molecules associate. Evidence against the hydrogen bond as a major cont r ibu t ion to the free energy of the pro te in-pro te in in terac t ion has been obtained lhe rmodynamica l ly [82]. On the other hand, van der Waals in teract ions , though they are more numerous as they involve all the pai r of ne ighbour ing atoms, are much less energe- tic and their overall con t r ibu t ion is small. The hydrophob ic con t r ibu t ion is largely dependen t upon the en t ropy gained by water due to the smaller accessible pro te in surface area when the pro te in molecules form a complex. The hydrophobic in te rac t ion seems to be ent i rely unspecific as compared to the complementar i ty of the sur- faces involv ing hydrogen bonds and van der Waals contacts ; ho~,ever, they decide which proteins can recognize each other.

V . T H E S P E C I F I C I T Y O F T H E A D S O R P T I O N

O F B I O M O L E C U L E S O N H Y D R O P H O B I C S U P P O R T S .

other, the ordered water molecules wil l be excluded and 'will adopt the less ordered buIk water state wh ich is equivalent to an increase in entropy. This hypothesis is supported exper imenta l ly by the study of an t igen-an t ibody complex forma- t ion [27] where a relative insens i t iv i ty to disso- lut ion of a preformed ant igen-ant ibody precipi ta te is observed, suggesting that once the complex is formed, the solvent is largely excluded in lhe regions of contact.

Thermodynamica l ly , the free energy value AG of a hydrophob ic in te rac t ion is a funct ion of AH and AS, accord ing to equat ion :

AG ~ AH - - TAS . . . . . . . . . . . . . . . . . . . . . (N ° 1)

Since AH is small as compared to TAS value, the process is fundamenta l ly de termined by the

Biospecific affinity, whe ther involv ing an << active site >> or not, p resumably depends to a large extent on the complementar i ty of the con- tours of the in te rac t ing molecules. In the absence of any specific effect, the b ind ing of prote ins to substi tuted agaroses is greatly affected by the overall charge of the biomolecule [105].

The lack of act ivi ty in the adsorbed state of some proteolyt ic enzymes [90], indicates that the b i nd i ng site is occupied by the hydrophob ic group of the subst i tuted agarose. Whether the hydrophob ic group interacts wi th the hydrophobic poc1~ets of the active sites of the enzymes or acts by a less specific ~way can be quest ioned. In any event, both cases may contr ibute to the adsorpt ion phenomena.

BIOCHIMIE, 1978, 60, n ° 1.

H y d r o p h o b i c ( in terac t ion) c h r o m a t o g r a p h y .

In other examples, the addi t ion of the specific substrate accompany ing the enzyme dur ing the chromatography (to mask the specific b ind ing sites) did not alter the b i n d i n g of the enzyme to the hydrophob ic matr ix, ind ica t ing that the adsorpt ion takes place through sites other than the specific substrate b ind ing site [971.

It is in te res t ing to note that g lutamine synthetase and other three prote ins involved in the regu- lat ion of glutamine metabol ism are all re ta ined by the same amino-aIkyl agarose derivat ives [99~ (see table II). Although, as Shaltiel and coworkers have signaled, this could be fortuitous, it might reflect a mutual hiospecific affinity among these prote ins s ince they must in teract wi th each other in order to effect their regulatory funct ions in the highly integrated glutamine synthetase cascade system.

The models proposed by Shaltiel [11] and Jennissen [78] explain in a different m a n n e r the mechanism by wh ich the in terac t ion between the prote in and the adsorbent occurs. For neutra l supports, Jennissen [86~ has presented evidence in favor of the idea that the adsorpt ion of proteins to aIkyl-agarose derivatives takes place at a cr i t ical alkyl group densi ty and is a funct ion of its hydrophobic i ty . In other ~vords, this hypothesis considers that the pro te in needs to present mul t iple a t tachment points in order to be adsorbed by a de terminate member of the series of alkyl-agarose derivatives. This is in agreement wi th an earl ier observat ion made by Hjert6n el al. [521. Shaltiel I l l on the other hand, suggests that the adsorpt ion of the prote in is due to the inter.action of an allkyl residue of specific length (¢ yard-s t ick ~) wi th a hydrophobic pocket of the protein. Evident ly , and as compared to Jennissen ' s model, the tatter implies a more specific mechanism. Ho~vever, it has been demonstra ted [583 that at high chain length, when the in terac t ions are stronger, the b ind ing is also less specific.

The <<positive cooperative in terac t ion >> [841 through the mul t iva lent b ind ing , may explain the increased free energy value of adsorpt ion of a de terminate member of the alkyl-agarose series. That is, a given prote in may be adsorbed by a cer ta in member of agarose-substi tuted series only if the number of ¢ contacts ~> is big enough to effect its re tent ion, and this can be done by varia- t ion of the degree of subst i tut ion. Comparat ively, amino-a lkyl agaroses present a << negative cooperative >> effect to,wards the adsorpt ion of phosphorylase b, possibly due to a different mechan i sm of in te rac t ion 'which does not necessar i ly exclude the mul t iva lent a t tachment E84].

It has been observed, in the case of some hydrophobic aIkyl- and a lkylamino supports, that cer- tain << s t rong >> b i nd i ng sites are occupied first, and that others of decreasing affinity become occupied when more protein is fed to the column. This is shown by the fact that only par t of the prote in can be eluted by increas ing the salt concentra t ion, whereas the remained is dislodged by the addi t ion to the eluant of a polar i ty reduc ing

{a) (b)

(c) (d) Fro. 2. - - Models of adsorption o[ proteins on hydro-

phobic matrices : a} The model suggested by Shaltiel [11] ; b) and c) Represents the two possibilities of mul- ti-attachment adsorption : on the matrix surface (b) and on a cavity in the matrix (c) ; d) The irregularity of the surface (attributed to the nature of the agarose structure) is the probable cause for 'which binding sites are not identical, and consequently the forces involved in the binding are different.

agent such as ethylene-glycol. It should be emphasized that this p rob lem is mostly related to those hydrophob ic supports which possess both hydrophobic and ionic groups, resul t ing from the cou- p l ing [70, 471 procedure or the nature of the l igand [96], like in the case of amino-alkyl derivatives. This apparen t inhomogenei ty of both mat r ix and prote in can lead to possible wrong in te rpre ta t ions about the pur i ty and characte- r ist ics of many prote ins [98, 105]. For instance, the recovery of rechromatographed materials is improved up to 95 per cent, as compared to that of 70 per cent obtained when the crude extract is applied into the column [98]. In other cases, desorpt ion of purif ied mater ial requires the va- r ia t ion of the eluant, as pointed above. It seems

BIOCHIMIE, 1978, 60, n ° 1.

6 J.-L. Ochoa.

TABLE II.

Hydrophobic matrices.

Type Coupling procedure Bepresentation References

I. Agarose derivatives

1. Un-substituted

2. Amino-alkyl-

3, Alkyl-

4. Amino acid

5. Other derivatives Fa t ty acids

AcetyI-N-amino-alkyl

Aniline Benzyl-ether Phcnyl-N-butyl-amine- NAD +- Hydroxy- alky 1 Diverse aromatic-alkyl derivatives

II. Dextran derivatives (Sephadex)

1. Acetyl-Sephadex

2. Methyl ether-(S)

3. Hydroxy-propyl- ether- (S)

4. Hydroxy-alkyL(S)

5. LH-20-(S)

I I I . Cellulose derivatives

1. Diethyl amino ethyl cellulose

2. Carboxy methyl-(C)

3. Benzoylated-DEAE-(C)

4. Esters of alkyl and aryl- (C) (paper, cotton)

IV. Glass derivatives

1. Alkyl-silated-(G)

2. Propyl_lipoamide-(G)

BIOCHIMIE, 1978, 60, n ° 1.

BrCN

BrCN

glycidyl ether

BrCN

BrCN (azide) BrCN (cvrbodiimide) (acetylation of amino-

alkyl-(A))

BrCN and glycidyl ether glycidyl ether

acetylation

acetylation

from commercial sources



benzoyl chloride

phenoxy acetylation

amino-silanization and amide bond formation with the corresponding alkyl chloride

(A) (A)-CH-N H-(CH~)n-N H,_,

I NH.~ +

(A)-CH-NH-( CH.~)-CH:~ I

NH~ +

(A)-O-CH~-CH-CH~-O- [ (CH'2)n-CH3

OH

(A)-glycine (A)-valine (A)-leucine (A)-tyrosine (A)-phenylalanine (A)-tryptophane

(A)-(CHs)n-OH

(s)

CH3-CO-(S)

CH3-O-(S) HO-CHs-CHs-CH.2-O-(S)

HO-(CH~)n-O-(S)

(C)

DEAE-(C)

CM-(C) BD-(C)

(G)

41, 47, li3] [9-11, 94, l i3, I l l ,

l i4, 58]

[II,94, I13, li4]

[52, l i2 l

[97, I15] [54, 68, 97, l i6, 77] [77, 97] [97] [35, 40, 42] [I051

llo6] 179, 58] [57, 94, li7]

[40] [93, 90] [105l [57, 58] [58, li2] [I12, 55]

H3ol [91] [9t]

[91] [9i]

[47, 71]

[47, 71]

[I03]

[101]

[ii8] [i07]

Hydrophobic (inleraction) chromatography.

TABLE II.

Type Coupling procedure Representation Relerences

V. Others supports

1. Polyamino methyl- styrene (polystyrene, polyamide, Dowex I-X8)

2. Alkyl-amine of poly- acrylic resins (butyl, capryl, lauryl, palmityl, steatyl, oleyl, linoleyl)

f rom commerc ia l sources 17t1

chlorination for amide [761 bond formation

that this d i sc repancy depends on the hydropho- bici ty of the prote in and the adsorbent , and moreover, that the b i n d i n g sites are not ident ical in a given car r ie r (which is true for most of the adsorbents employed in chromatography in general).

The mult iple po in t a t tachment b ind ing is most l ikely to occur in a cavity of the adsorbent than on its surface, pa r t i cu la r ly when the pro te in molecule fits into the cavity (fig. 2). Conversely, at points on the matr ix ~vhere the bound l igands are dis t r ibuted over a p ro t rud ing area, b ind ing would he less strong. S i n c e the surface of the adsorbent may be assumed to be irregular , many different s i tuat ions in addi t ion to these two hypothet ica l cases ~vould be obtained. Addit ional ly , small amounts of pro te in b ind more homogeneously on a par t icu la r column than a large amount. This could mean that by reduc ing the amount of appl ied protein, the s t ronger b i n d i n g sites might p redomina te in the b inding.

If mul t iple point a t tachment were one of the reasons for s trong non-specific adsorpt ion, one ~vay to reduce it ~vould be to louver the degree of subst i tut ion to the point where the distance between the subst i tut ion groups is larger than the diameter of the prote in molecule. This ~vould not affect the specific ¢ one-to-one >> in terac t ion as that between an enzyme active site and an immo- bilized subslrate analogue.

VI. THE FACTORS INVOLVED IN HYDROPHOBIC (INTERACTION) CHROMATOGRAPHY.

A. The matrix hydrophobicity.

1. Matrices and supports.

Proteins differ in their hydrophobic i ty as a funct ion of their p r imary structure, that is, in the

BIOCHIMIE, 1978, 60, n ° 1.

sequence and amino acid composit ion. Hence, it is not surpr i s ing that the first type of hydrophobic supports ever employed were derivated from coupl ing various non-polar amino acids to an iner t support or matr ix like agarose [54]. Other supports have also been e m p l o y e d : cellulose, glass, dextran, etc. (see table II), but the ideal matr ix 'without secondary non-biospecific adsorp- t ion effects has not been found.

All the different types of supports and matrices util ized at the present possess undes i rable interfe- rences a t t r ibuted to their chemical composit ion. Addi t ional effects arc obtained in such away that is difficult to obtain a pure hydrophobic interac- t ion chromatography after the in t roduc t ion of the spacer-arm or hydrophobic ligand.

In table II, the various types of matrices employed in HIC, as well as the different types of l igands coupled, have been summarized. Refe- rences are given to i l lustrate specific applicat ions.

As has been demonstrated, un-subst i tu ted agarose is sufficiently non-polar p resumably due to the 3-6 methylene diether bridges present in every second galactose residue of the polysacchar ide chain [67] with respect to lhe re tent ion of halophi l ie prote ins [47] and nucleic acids [41, 119], at high salt concent ra t ions (2.5 M a m m o n i u m sulfate). Though the halophi l ic proteins are highly negatively charged [48], as a result of an excess of acidic groups [49], it is believed that the high salt concent ra t ion el iminates the possible ionic interact ions.

When DEAE- and CM-derivatives, either of cellulose or agarose, have been used, the elution pat tern shifts to higher or louver salt concentra- t ions than those required 'when tile neutral deri- vative is used as a support. This is explained by

8 J . -L. Ochoa.

e lec t ros ta t ic a t t r ac t ion - repu l s ion forces be tween the negat ive ly charged ha lop ro te ins and the nature of the charge on the matr ices .

Wi th few except ions , agarose is the type of ma t r i x most commonly used (table II), and the k ind of l igands or space r - a rm in t roduced are in a w i d e range of h y d r o p h o b i c i t i e s and s t ructures . Consequent ly , the h y d r o p h o b i c i t y of the nmtr ix depends on the type of the l igand E78], and on the degree of subst i tu t ion [5,6, 86]. In this respect , the p rope r t i e s of the l igand should be d iscussed first.

2. The role of the l igand in the h y d r o p h o b i c i t y of the mat r ix .

As po in ted out above, HIC was born (in many cases) as a consequence of non-biospeci f ic adsorp- t ion found in aff ini ty c h r o m a t o g r a p h y systems and a t t r ibu ted to the type of space r - a rm used to b r idge the l igand to the mat r ix . Nevertheless , evi- dences exis t that p rove that HIC was conce ived by Hjer l6n and his group in a d i f ferent way, whi l e s tudy ing the so lubi l iza t ion of membrane pro te ins . They a t t empted the i r s epa ra t ion us ing a h y d r o p h o b i c suppor t to 'which a de te rgent (SDS) was coupled. In th is case, however , the adso rp t ion was so s t rong that the p ro te ins could not be deso rbed by any non-dena tu r ing system (unpubl i shed results , pe r sona l communica t ion ) . When Cuatrecasas [87] sho~,ed that ~-galactosidase could only be f rac t iona ted if the l igand was suff ic ient ly sepa ra t ed from the mat r ix , and thus avo id ing s te r ica l h ind rance , the idea of separa- t ing l igands f rom the ma t r ix backbone by use of space r -a rms r a p i d l y spread . Very soon, it became clear, that m a n y p ro te ins were adso rbed non- speci f ica l ly because the p resence of the arm int roduced non-biospeci f ic adso rp t ion centers . F o r ins tance , a space r - a rm c a r r y i n g charged groups gives or ig in to e lec t ros ta t ic in te rac t ions [106]. The subs t i tu t ion of the cha rged arms by o ther neu t ra l chemica l analogs was thought to be an excel lent a l t e rna t ive but then, o ther type of non-specif ic in te rac t ions , h y d r o p h o b i c in nature , tu rned out to be impor t an t [7].

Yon [9], Er-el et al. [10] found that p ro t e in could b ind subst i tu ted agarose mat r ices w i thou t any specif ic l igand. The adso rp t ion ~ras a t t r ibu ted to the p resence of the d iamino-a lky l group employed as a spacer . This observa t ions were fu r the r suppo r t ed by O 'Carra and coworke r s [57, 88], Who found that the p resence of the l igand d id not al 'ways account for the r a t a r d a t i o n effect and again the space r - a rm was cons ide red as respons ib le . Yon [9] suggested that it was poss ib le to take advantage of such non-biospeci f ic adsorp-

t ions and showed that ce r ta in p ro t e ins could be se lec t ive ly adso rbed on decyl -agarose der iva t ives d i f fer ing in the ionic cha rac t e r of the spacer -a rm. Later , Shal t ie l and his group [10, 11] r e p o r t e d pur i f ica t ion of severa l enzymes on a ser ies of a lkyl and amino-a lky l agarose der ivat ives . Almost s imul taneous ly , Hjert6n and his group [521 repor ted , for the first t ime, the p r e p a r a t i o n of diffe- ren t subs t i tu ted agaroses 'with uncha rged groups that could i l lus t ra te an exclusive h y d r o p h o b i c mechan i sm of adsorp t ion .

The effect of the h y d r o p h o b i c i t y of the spacer - a rm in the pur i f i ca t ion of var ious p ro t e ins [8-11] has been poss ib le thanks to the deve lopmen t of the (< mock aff ini ty systems >>. Steers [87 demons t ra t ed that the adso rp t ion of ~-galactosidase was s t rongly re la ted to the length of the spacer - arm. The series of aIkyl and amino-a lky l der iva- t ives of agarose p r e p a r e d by Shal t ie l have also been successfu l ly app l i ed in m a n y cases [11].

The d i f ference of a s ingle C-atom in agarose bound N-aIkyl groups may have a large effect in the h y d r o p h o b i c b ind ing of a p a r t i c u l a r p ro t e in [75]. There fo re i n t e r m e d i a r y h y d r o p h o b i c compounds be tween two consecut ive member s of a homologous ser ies of l igauds are needed. Such in t e rmed ia t e h y d r o p h o b i c i t i e s can be obta ined , for ins tance, t h rough the i n t roduc t ion of a cha rged or o ther h y d r o p h i l i c group, e.g., hydro - xyl group. The i n t roduc t ion of double bonds or of b r a n c h i n g of the chain , reduces also t he hyd ro - phob ic i t y as c o m p a r e d to the c o r r e s p o n d i n g sa- tu ra ted s t ra igh t cha ins [13]. Weiss and Bucher ['76] have p r e p a r e d some a lkyl agarose der iva t ives of i nc reas ing insa tu ra t ion for this purpose . In add i t i on a roma t i c der iva t ives may possess in ter - media te h y d r o p h o b i c i t i e s be tween t~wo consecu- tive members of the a lkyl -agarose series. Benzene, for example , is equiva lent to that of 3-4 s t ra ight cha in h y d r o c a r b o n [19].

Al though Tanfo rd [1O] has showed that the h y d r o p h o b i c i t y of a l inea r a l ipha t i c carbon increases l i nea r ly w i t h inc reas ing number of CH 2- groups, Shanbhag [50] cons iders tha t the effec- t ive h y d r o p h o b i c i t y depends also on the f lexibi- l i ty of the h y d r o c a r b o n cha in and consequent ly on the degree of in te rac t ion w i th in such chains , spec ia l ly for long chains. Hofstee [75], on the o ther hand, assures that ne i the r the d i f ference in molecu la r shape of the N-alkyl l igands and the s ide chain of a romat ic compounds l ike phenyl - a lan ine or t r yp tophan , nor the d i f ference in net charge of the adsorben t is a d e t e r m i n i n g fac tor for p ro te in b ind ing . It seems then, that the me-

BIOCHIMIE, 1978, 60, n ° 1.

Hydrophobic (interaction) chromatography.

chan i sm of the adso rp t ion of p ro te ins to hyd ro - phob ic ma t r i ces fol lows some very complex rules. This p r o b l e m can be exempl i f ied by the case of the adso rp t ion of e ry th rocy te s on a ser ies of aIkyl -agarose der iva t ives [110] in ~which case a decrease in adso rp t ion bet 'ween C6-C s is no t iced to occur wi thou t a reasonable exp lana t ion .

The combina t ion of the p r inc ip l e s of ionic- exchange c h r o m a t o g r a p h y and HIC has been successful ly app l i ed in some cases [9, 41]. The use of l igands con ta in ing both ionic and h y d r o p h o b i c cha rac t e r s have been r e c o m m e n d e d in o rde r to avoid the dena tu r ing effect de tec ted in the use of a lky l -agaroses [108]. It was also argued that the elut ion p r o c e d u r e might be mi lde r than in the case of pure h y d r o p h o b i c space r -a rms or l igands [9] and fu r the rmore , that the use of charged arms was capab le of f iner d i s c r imina t i on be tween l i poph i l i c pro te ins . The pro te in , in this case, in te rac t s h y d r o p h o b i c a l l y , involv ing the a lkyl chains , and e lec t ros ta t ica l ly , involv ing the ter- mina l ionic groups. W i t h this type of adsorbents , if the c h r o m a t o g r a p h y is ca r r i ed out at a pH equivalent to the i soe lec t r ic po in t (IP) of the protein, the adsorp t ion wi l l be due to h y d r o p h o b i c b ind ing alone. By changing the pH to in t roduce in the p ro te in a net charge of the same sign as the charge of the adsorbent , the r epu l s ion effect wi l l decrease the adsorp t ive force due to h y d r o p h o b i e bonding, mak ing poss ib le the desorp t ion . Obviou- sly, a dra~vback in us ing this type of adso rben t ar ises f rom the p rob l em that f requent ly the IP of the b iomolecu le of in teres t is unknown, and that many p ro te ins p r ec ip i t a t e at the i r IP. Moreover , the s imul taneous p resence of ex t raneous ionic and h y d r o p h o b i c groups in aff ini ty adsorbents , has been found to cause a subs tan t ia l non-specif ic p ro te in b ind ing thus resu l t ing in a r educed bio- spec i f ic i ty of these mate r ia l s [106].

3. The inf luence of the degree of subs t i tu t ion on the h y d r o p h o b i c i t y of the mat r ix .

The degree of subs t i tu t ion on Sepharose 4B w i t h a Ikyl -amines of di f ferent h y d r o p h o b i c i t i e s is a c r i t i ca l p a r a m e t e r in the adso rp t ion of p ro- teins. About 1012 a lkyl res idues pe r Sepharose sphere appea r s to be a c r i t i ca l degree of subst i- tut ion for the adso rp t ion of enzymes l ike phospho ry l a se k inase , phospho ry l a se phospha tase , 3'5'-cAMP dependen t p ro te in k inase , g lycogen synthetase , and phospho ry l a se b w h i c h are suc- cess ively adsorbed when the h y d r o p h o b i c i t y of the Sepharose is increased . In addi t ion , the degree of subst i tu t ion de te rmines the capac i ty of the gel [78].

The c r i t i ca l h y d r o p h o b i c i t y needed to adsorb p ro te ins can be ob ta ined by e i ther inc reas ing the degree of subs t i tu t ion or by e longat ing the employed a lky l -amine chain at a cons tant degree of subst i tu t ion. Consequent ly , as the h y d r o p h o - b ic i ty of the gel is increased , h ighe r b ind ing affi- ni t ies resul t and the deso rp t ion requi res more and more severe condi t ions . In the case of neu t ra l a lky l -agaroses [3~1], e lut ion of p ro te ins f rom the h y d r o p h o b i c ma t r i x can be desc r ibed in te rms of sal t ing- in phenomena , s ince desorp t ion is dependent on the type of salt employed and not on the ionic s t rength alone.

In all cases, a min ima l cha in length seems to be r equ i r ed in o rde r to obta in the adso rp t ion of a given pro te in . This fact has been i n t e rp re t ed as f i t t ing a h y d r o p h o b i c group into a h y d r o p h o b i c pocke t of the pro te in . By var ia t ion of the concen- t ra t ion of cyanogen b r o m i d e in the ac t iva t ion mixture , the amount of h y d r o p h o b i c res idues may be v a r i e d . Thus, the c o r r e s p o n d i n g hyd ro - p h o b i c i t y may be inc reased such that the amount of adso rbed mate r i a l inc reases exponen t i a l ly when the degree of subs t i tu t ion of the gel is enhanced . Control expe r imen t s w i th methyl -agarose show that s imi l a r ly inc reased degrees of subst i tu t ion, and thus s imi la r numbers of pos i t ive charges i n t roduc e d by the coupl ing p r o c e d u r e [117], do not affect the adso rp t ion of the assayed pro te in . Therefore , the adso rp t ion of l a rger amounts of mater ia l , de t e rmined by inc reas ing the degree of subst i tu t ion, is not a funct ion of the add i t i ona l number of charges [78]. One may conc lude that, if in a ser ies of gels of d i f ferent h y d r o p h o b i c i t i e s a c rude ex t rac t con ta in ing hy- d r o p h o b i c a l l y d i f fer ing p ro te ins is appl ied , the one vcith the h ighes t h y d r o p h o b i c i t y wi l l be adsorbed by the gel of the lowest degree of subst i tu- t ion. Then as the number of a lkyl res idues increases on the ma t r ix , p ro te ins of lower h y d r o - phob ic i t i e s are adsorbed .

A d i rec t me thod of de t e rmin ing the degree of subs t i tu t ion for charged h y d r o p h o b i e groups has been p r o p o s e d by Hofstee [89]. The method des- c r ibes the use of Ponceau S, a dye w h i c h ca r r i e s h y d r o p h o b i c groups in conjunc t ion w i th an overa l l negat ive charge bound in an i r r eve r s ib l e fashion to the l igand. Under a given set of condi - t ions, and af ter app l i ca t ion of a sa tu ra t ing amount of dye, a cer ta in amount wi l l r ema in bound even af ter extens ive wash ing . Eighty-five pe rcen t of the Ponceau S b ind ing capac i ty is lost af ter a pe r iod of a lmost 5 months i nd i ca t i ng that the degree of subst i tu t ion decreases g radua l ly upon storage. In some cases, 40 pe r cent was lost in only 40 days.

BIOCHIMIE, 1978, 60, n ° 1.

1 0 J.-L. Ochoa.

The data also suggest that the adsorbents wi th the highest degree of subst i tut ion are the least stable [89].

An est imation of the relative degree of substi- tut ion can be obtained from the acid-base treat- ment of the adsorbent [89], by a nucleophi l ic at- tack of N-substi tuted isourea (derivated from the coupl ing procedure 'with BrCN) wi th an active chromogenic substance [70] ; or more indirect ly , by measur ing the capacity of the adsorbent wi th a coloured protein like cytochrome C [90] or phy- coery thr in [56]. A inuch more sophist icated but accurate method utilizes NMR spectra [56].

Final ly , it is impor tan t to ment ion that a consequence of the degree of subst i tut ion is the shr inkage of the gel owing to a decrease in hydrophi - l ici ty due to the hydrophobic character of the ligand. Depending on the special s t ructure of the agarose gel, the shr inkage seems to be much less impor tan t than wi th other gels l ike Sephadex or po lyacry lamide [90].

B. The influence of salt.

The influence of salt on the adsorpt ion of proteins to hydrophob ic supports is p robably due to a number of factors act ing on the prote in as well as on the matrix. It has been demonstra ted that neutra l salts induce conformat ional and s t ructural changes in biomolecules [34]. These studies have been carr ied out by c i rcular di- chroism spectra of the protein at cons,taut ionic strength. It has been concluded that salting-out ions cause conformat ional but not s t ructural changes whereas salt ing-in ions cause sometimes severe s t ructural changes wh ich may be one reason of their dena tu r ing effect [34].

The << s tructure forming >> propert ies of salting- out ions enhance in t ramolecular , as wel l as inter- molecular , hydrophob ic bond ing as reflected by a stabil izat ion of the hydrophob ic core of the biomolecule [35, 43']. The effect of specific ions on macromolecules was first noticed by Hofmeister [36]. He found that salts differ greatly in their abil i ty to salt-out prote ins at a given salt concen- trat ion. At h i g h concent ra t ions of salt ing-out ions, the solubi l i ty of the prote in is adversely affected by decreasing the avai labi l i ty of ~vater molecules in the bulk and increas ing the surface tens ion of water, resul t ing in an enhancemen t of the hydrophobic in teract ions [31, 371. Accordingly, the influence of neutra l salts on the adsorpt ion phenomena determines to a large extent the degree of adsorpt ion and correlates closely wi th the Hof- meister series [63, 58]. Salt ing-in ions, on the other

BIOCHIMIE, 1978, 60, n ° 1.

hand, are << s t ruc ture-breaking >> ions, and thus do not favour hydrophob ic interact ions. This effect can be regarded as an inevi table consequence of the new order imposed by the ion or ient ing water molecules in such a way that water cannot undergo fur ther positive entropy change, as it is required for the formation of hydrophobic bonds [18]. These ions have also been termed ¢ chaotropic >> [44] because they provoke unfolding, extension and dissociat ion of the macromolecules. In this respect, they might be used in the elution of s trongly adsorbed materials on hydrophob ic supports.

Already in 1948, Tiselius [38] noticed that proteins and other substances (e.g., dyes) that could be precipi ta ted by high concent ra t ions of neutral salts could be adsorbed (at much lo'wer salt concentra t ion) to common adsorbents , whereas in the absence of salts those adsorbents showed no affi- ni ty for the substance. In the years afler~vards, some attempts have been made to pur i fy prote ins on solid supports us ing gradients of ammon ium sulfate [39, 40, 54]. In general, proteins which pre- cipitate at low ammonium sulfate concent ra t ions should l ikewise be re ta ined on hydrophob ic supports at low salt concent ra t ions ; and similarly, prote ins ~vhich precipi ta te at high concent ra t ions of a m m o n i u m sulfate would require relatively higher concent ra t ions of salt to obtain their reten- t ion [40].

The purif icat ion of nucleic acids [41] has also been reported using unsubst i tu ted agarose with high concent ra t ions of ammon ium sulfate. It should be emphasized that tbe adsorpt ion occurs at concent ra t ions below which the macromolecules precipi ta te out of solution. Since the adsorption is control led by salt concent ra t ion , ra ther than by the hydrophob ic i ty of the column, the names of << salt-mediated hydrophobic chromatography >> and hydrophobic (salting-out) chromatography have been employed elsewhere [42, 54. 67]. Hovccver, the hydrophob ic i ty of the gel and the effect of the salt concent ra t ion may be combi- ned efficiently to improve the adsorpt ion phenomena a n d / o r the puri f icat ion [64J, especially of prote ins wh ich may be affected by the high salt concent ra t ion required for its adsorpt ion on the given gel [46]. If the use of high salt concentra- t ions is l imited by the enzyme activity or s tabil i ty for instance, a compara t ive ly longer a lkyl-chain must be employed in order to obtain sufficient capacity. Because a s imi lar sa l t ing-out /sa l t ing- in effect has been noticed in the case of high concentra t ions of sugars, namely sugaring-out and sugaring-in effect [45], the possibi l i ty in using


sugars instead of salts should be considered. Ho- wever, this effect is p robably not general.

C. Effect of temperature and pH.

From the equat ion N ° 1, it is clear that the temperature may influence, in a posit ive way, the adsorpt ion of prote ins on hydrophobic matr ices ~52, 551. However, tempera ture may also provoke other effects, such as increased solvation and decreased surface tension, ~vhich may wi ths tand the abi l i ty of molecules like prote ins to in terac t xvith hydrophobic supports [53J.

The prote in molecules re ta in their biological ac.fivity or capaci ty to funct ion only w i th in a l imited range of tempera ture and pH. Thei r expo- sure to extremes of pH and temperature causes them to undergo dena tura t ion ; the most visible effect is a decreased solubil i ty of globular proteins. Since the covalent chemical bonds in the peptide backbone of the prote in are not broken dur ing denatura t ion, it has been concluded that it is due to the unfo ld ing of the character is t ical conformat ion of the native form of the protein molecule. The refolding of a denatured protein does not require the input of chemical work from outside. It proceeds spontaneously, provided that the condi t ions of temperature and pH are adjusted to be compatible wi th the s tabi l i ty of the nat ive conformat ion of the protein. In this respect, the process is s imilar to the one in hydrophob ic bond formation.

a l ter ing its te r t iary s t ructure [65] and provoking its desorpt ion from the charged alkyl amine agarose columns. On the other hand, b ind ing at pH 7.5 does not occur probably due to other conformat ional changes in the molecule, which could mask key sites involved in the b i nd i ng E41_~.

Lysozyme, a basic protein, becomes more and more re tarded on Clo (aIkyl Sepharose) as the pH of the eluant is lowered, and b inds to this co lumn at pH 1.5 [661. Cytochrome C is also highly pH- dependent in its adsorpt ion to neutral ethers of agarose derivatives [67J.

No influence of pH has been observed on the adsorpt ion of polysacchar ides to subst i tuted agaroses [68~. In this case, the re tent ion is mostly de te rmined by the hydrophob ic i ty of the column and by the size of the polymer.

Naturally, pH plays an impor tan t role in the adsorpt ion of biomolecules on hydrophobic matrices ca r ry ing charged groups. With this type of adsorbents , pH may modify the charge either of the biomolecule or of the adsorbent . By changing the pH, a net charge may be intro- duced in protein, wh ich can be of the same sign, or opposite of the charge on the adsorbent . In the first case, the repuls ion effect ~vill affect nega- t ively the adsorpt ion phenomena. As was pointed out previously, a maximal hydrophob ic adsorp- t ion wil l thus occur at the isoelectric point of the prote in E9!.

The s t rength of hydrophob ic bonds should increase wi th r is ing tempera ture up to above 60°C, at wh ich point the addi t ional stabil i ty ar is ing from hydrogen bonding, electrostatic forces between charges or dipoles, van der Waals interact ions and disulfide bridges disappear, and turn against the favoured hydrophob ic in terac t ion [18, 30, 50] of the protein.

A pH effect on hydrophob ic bond ing is observed in the case, for example, of bovine serum al- bumin b ind ing alkanes at pH 4. It 'was observed that at low pH the site of hydrophob ic bond ing is disrupted [61J, and that the degree of aggregation of the globulin does not affect the degree of binding. The conclusion ~vas that the sites associated wi th aggregation are relat ively non-hydrophob ic and that any conformat ional changes, resul t ing from the polymer iza t ion do not exert any effect at the hydrophob ic b ind ing site of alkanes [62].

Decreasing pH belo'w pK value of the amino group of adenyl ic and cyt idyl ic acid residues of tRNA changes the negative charge of the molecule,

VII. METHODS OF DESORPTIOX on ELUTION OF ADSORBED MATERIALS ON HYDROPHOBICS MATRICES.

The elution of adsorbed materials from hydrophobic gels can be obtained in a number of ~vays depending on the type of adsorbent employed, the condi t ions in 'which the adsorpt ion occurred, and the propert ies of the biomolecule.

When proteins have been adsorbed at high salt concentra t ions , a decreasing ionic s trength of the eluant ~vill usual ly result in the removal of the mater ial f rom neut ra l adsorbents [71, 791. If the support carries charges in t roduced either by the coupl ing procedure [70], or by the nature of the l igand, electrostatic in terac t ions wil l become impor tan t at low ionic strength, and the elution wi l l not be possible [47, 1031. In such cases, changes in the pH [9, 65], buffer composi- t ion [47], temperature , or the addi t ion of non- polar organie substances may be quite useful procedures.

BIOCHIMIE, 1978, 60, n ° 1.

12 J.-L. Ochoa.

The fact that in many cases elution is possible by ra is ing salt concent ra t ion , demonstrates the presence of cooperative electrostatic in te rac t ions in the overall adsorpt ion phenomena in the case of charged-hydrophobic supports. Nevertheless, it has been shaw that in those si tuations, the mechan ism of hydrophob ic adsorpt ion at high salt concen t ra t ion prevents the electrostatic interact ions of be ing the main dr iv ing force involved in the process.

The use of dena tu r ing agents, such as urea, guanidine-HC1 etc, has been successfully applied in desorpt ion procedures. Thei r mechanism of act ion appears to be involved in both direct interaction 'with the s ide-chains and peptide groups and d is rupt ion of hydrophob ic in terac t ions between side chains [23, 69, 77, 109].

Chaotropie ions, on the other hand, are know n for their abi l i ty to impar t l ipophi l ic propert ies to water [72] and to alter the s t ructure of biomolecules [44]. Thei r appl ica t ion for the elution of prote ins from high members of alkyl-agarose de- r ivatives has been reported to be successful [73] ; ho'wever, it is impor tan t to bear in m i n d their p roper ty of dissolving agarose gels and denatura t ing proteins. In this case, cross-l inked agaroses, wh ich are kno'~vn to support more severe condi t ions (pH, temperature, ionic strength etc) than the normal agarose gel, are to be employed.

As has been men t ionned before, by increas ing the hydrophob ic i ty of the gel t ighter b ind i ng re- suits, and desorpt ion of prote ins requires ever more drast ic condi t ions [78]. The combina t ion of salt gradients wi th << polar i ty- lower ing >> agents (e.g., ethylene glycol):, var ia t ion in pH or temperature, etc., may give an increased selectivity of the elution. As an i l lustrat ion, it can be ment ionned the case of serum a lbumin, where elution occurs at pH 3 wi th 50 per cent ethanol in the buffer [791. Similarly, the use of mixed gradients of buffer solution wi th high salt concent ra t ion , and buffer wi thout salt but con ta in ing 50 per cent of ethylene glycol, have been successfully appl ied to desorb enzymes such as chymot ryps in and t ryps in from hydrophob ic matr ices [90].

A simple pure salt gradient may result in a s trongly diluted peak in the elution because the cond,itions in 'which the equ i l ib r ium for the hydrophob ic in te rac t ion is at tained are slob,. Only lower flow rates could provide more favou- rable results. In addi t ion, it should be emphasized that in some cases the elution with ethylene glycol, in the absence of salts, may be inefficient due to the presence of electroslatie in te rac t ions

[98] w h e n adsorbents involv ing mixed effects are employed.

The use of detergents [127] is usually the last resource, w h e n the elution by other mi lder condi- t ions has not been possible. The problem in using detergents is their in t r ins ic dena tu r ing effect and the difficulty of their removal from the columns dur ing the regenerat ion step [127]. A wide range of different detergents has been used vary ing in their efficiency to el iminate strongly adsorbed materials E127]. In this respect, the more ionic ones are to be preferred for the reasons discussed above.

Final ly , flat curves are obtained by gradient elut ion [75], p resumably due to a postulated i r regular i ty of the matr ix (see fig. 2) and the occurence of a wide range of b ind ing sites of varied strengths [74]. For this reason, attempts should be made to fract ionate through << different ia l -adsorpt ion >> [123] (as opposed to different ia l -elul ion) on a series of adsorbents of increas ing hydrophobic i t ies . In this way, each prote in tends to be adsorbed or bound to the column that provides the m i n i m u m degree of hydrophob ic i ty requi red for b inding , and com- plete elut ion of the materials can be accomplished wi th mild eluants. Another al ternat ive consists of the use of a high member of the alkyl agarose series, which may retain a high percentage of the prote in content of a par t i cu la r mixture, and allows the elution of the molecule of interest unde r mild condi t ions nvhile most of the other pro te in remain adsorbed on the column [123]. Obviously, this possibi l i ty can be uti l ized only when the biomolecule in question has a lo~,- hydrophobic i ty . One should note that in p r inc ip le it should be possible to achieve puri f icat ion by HIC, not only by a selective re tent ion of a given protein, but also by exclusion of a desired prote in when most of the other prote ins in the mixture are adsorbed [114].

VIII. A P P L I C A T I O N S , F U T U R E AND IMPLICATIONS

OF HIC.

The relevance of HIC lies in the fact that it is perhaps the first technique w h i c h takes advantage of the hydrophob ic propert ies of the biomolecules.

The hydrophob ic character of biomolecules should be a specific proper ty , s imilar to the ionic characler , s ince this is a funct ion of the p r imary s t ructure in the case of prote ins and nucleic acids. So, it is not unreal is t ic to consider that

BIOCHIMIE, 1978, 60, n ° 1.

H y d r o p h o b i c ( i n t e r a c t i o n ) c h r o m a t o g r a p h y . 13

their selective separat ions are feasible. As an example, the pur i f icat ion of two in te rconver t ib le forms of glycogen-phosphorylase, wh ich differ in a un ique serine residue, has been successfully achieved through their abi l i ty to adsorb on two dis t inct members of the alkyl-agarose series [129].

Unfor tunate ly , the types and nature o f the hydrophobic adsorbents available to date are probably not well systematized, and the lack of inter- mediate hydrophob ic matr ices l imits the applica- bi l i ty of the method. To provide for a larger variety in the types of l igands, i nc lud ing the in t roduc t ion of aromatic structures, it might be expedient to at tach these l igands to (< arms )~ that by themselves are not hydTophobic, e.g., carbo- hydrates instead of hydroca rbons [96]. At first sight, hydrophobica l ly coated supports may be used as concen t ra t ing systems as well [52]. In fact, t h e prote in 'which is going to be purified does not require a p re -concent ra t ion step if the condi t ions unde r 'which t h e chromatography is performed allow its adsorpt ion.

The appl ica t ion of HIC to the pur i f icat ion of nucleic acids using matr ices of var ied hydropho- bici t ies according to the type of l igand at tached or to its degree of subsl i tu t ion awaits for fur ther studies which may greatly improve the applica- t ion of this technique. This aspect is especially in teres t ing when the molecules are not stable or soluble at ei ther too high or too low salt concen- trations.

The separat ion of part icles, like sub-cel lular fract ions or entire cells [110, 121] has been reported, and it seems to be a very p romis ing applica- t ion of HIC, s ince membranes can be considered as aggregates of molecules of var ied hydrophobi - cities. A s imi lar appl ica t ion in a quite different field of b iochemis t ry is p repara t ion of enzymat ic reactors through the immobi l iza t ion of enzymes on hydrophobic supports [32, 101, 122, 126]. The basic idea is the possibi l i ty to have a reactor wh ich can be per iodical ly recycled, when the adsorbed mater ial loses its act ivi ty by elution and adsorpt ion of freshly active substances (which may be of the same or of another biological activity, but s imilar ly adsorbed on the hydrophob ic support) . Thus, the hydrophob ic matr ix may funct ion as an universa l suppor t of easier hand l ing than those systems in wh ich the immobi l iza t ion necessitates a covalent l inkage between the biomolecule and the adsorbent , wi th their cor responding l imita t ions in funct ion and util i ty,

It should be reminded that some hydrophob ic l igands denature prote ins through a <( detergent-

like >> action [96]. This effect can be reduced by employing hydrophobic l igands of mi lder influence, or by in t roduc ing addi t ional polar or ionic groups in the hydroca rbon chain [9]. Fi- nally, the problem of << inhomogenei ty >>, wh ich consti tutes another impor tan t d rawback in prote in separat ion by chromatography on hydrophob ic columns, must be considered. If inhomogenei ty occurs in the adsorbent , that is if the in teract ing sites on a given member of the suhsti tuted-agarose series are different in their s trength of in terac t ion wi th a par t icu la r prote in (fig. 2), elut ion by decreasing salt concent ra t ion or increas ing concent ra t ion of << polar i ty- lower ing >> agents, wil l never result in a na r row peak but ra ther in flat curves, which increase con tamina t ion of the sam- ple by other proteins. Some suggestions to avoid this phenomenon have already been ment ioned taking advantage of the hydrophob ic i ty of the matrix.

In conclusion, the correct appl ica t ion of hydrophobic chromatography implies the considera t ion of three ma in factors :

- - the na ture and hydrophobic i ty of the adsorbent and of the protein,

- - the condi t ions of the adsorpt ion,

- - the condi t ions of the elution of the adsorbed material .

It may be expected that as the mel lmd wil l be more and more aplied wi'.h various biomolecules, in different condi t ions, our unde r s t and ing wil l be enr iched so about many biological events in which a hydrophob ic in terac t ion is involved.

Acknowledgments.

The author expresses his grat i tude to Dr. Jean-Marc Egly for his encouragement in the preparat ion of this article. Special thanks are due to Professor S h m u e l Shal t ie l and Professor Ste l lan Hjertdn for their cri- t ic ism and suggestions, and to Professor Barbarin Arreguin for his cont inuous interest on m y ,work. My appreciat ion to Dr. Dana Fawlkes for the l inguist ic reoision and to Mrs. Inga Johansson and Mr. GSsta ForMing for typing the manuscr ip and drawing the f igures, respectioely.

To the Nat ional Universi ty of Mexico and the Natio- nal Council of Sciences and Technology of Mexico m a n y thanks for their f inancia l support.

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