BZ~GEN 23~17

A n o m a l o u s p r o p e r t i e s o f w a t e r in m a c r o m o l e c u l a r gels John Van Stevenh~ck, M~he! P~rdekoo~r, and Ehud Ben-Hug

Tom M.A.R. Du~ehnan *

~ . In u a ~ . ~ - [ ~ m s stmlk~ this ~ ~ ~as ~ to rite ~ d" a t n m t ~ t m - r ~ lm~es ~m ~ ~e~ ~ ~ e n m ~e~. ~ . ~ e m c k ~ k s , m explain K,~ ~ l m ~ lmmr tl~m mtit~. Ka t a m e s 05 s m ~ ,a~mes l ~ n - tl~m tmit~ ~ere mmtll~ ~ t¢ adSmlm~m of the ~ l m e m ~ ~ ~ ~ ~ ~ tmper ~ ¢~,et~micus ~ ~ t h m ¢mmmlict these ~ e m ~ s . Eximimem~ ~ ~ ~ ~ - i u i water l ~

at the gel ~ x - w a t e r

~

.

so~oem ~

lntrmhxti~ S h o ~ - ~f~er the ~%'~clo~mem of d e ~

in which ~ is the elu~ion ~ofm~e of the sotute, ~ is the ~ d ~ofmne (the e l a t k ~ ~ e of m o ~ e s on b present in the mobihe phase, because they are larger than the largest p ~ e s in the gel). ~ is the ~ofume of

i ~ e the gel g ~ c ~ s : ~. to~ai ~ater ~i~m~ of coicmm I¢~. apparent mx'~N~entI ~ate~. DMSO. dimethy[suifox~de: PEG. pob~etMie~e gbxol~. Bk~:hem~h-3~.P.O. Box 9503. ~

is a ~ ! ~ e

to ~ y

ge~s f t ,

t e n s t ~ s ~ot m ~ o r ~ r ~ e ~ i t h their rm~e~k~r sine [2-8]. ld~aJb'. ~ h e n a ~ u t e ~ s s e s ¢kmn a gel bed. i~s m~ement s~houk~ ¢~epe~ on the b~d~k fio~- of ~he mobile phase and its diffusion into and oat of the soN~ent inside the pores of the stationary, phase. "~¢s. the eiut/o~ of a solute from a gee f ~ t r a t ~ ¢ o ~ can be characterf~ed bS. i ~ distn'but/on coefficient. Kd:

meat of M ~ Netheriamts.

the gel, ~ c h

RA L¢~e~.

~ a m e of the colamm A p p a r e n t ; file K~ ~ •- ~ - b e ~ ' e e n O a ~ L for k ~ e a ~ ~ e ~ sma~ respectively. Many small ~ : ¢ s . ho~-e~er, e x h ~ t a K j valme eitber km-er or h~gher than unity. From a point of ~ - t ~ ~peaed ~.he ]L~SS~ty to separate chem~ red,ted soh~es of low. equal m ~ h ~ r size on gel fdtration med~a [4.9]. From a the~efical point of view these obse~tions couM ~ be ¢~l~Med by ~ec~ar s ~ i n ~ . In mos~ smdhes K~ ~ u e s of s m ~ molecules io~-er than urfi~- ~-ere ~ n ~ e b " a~tn~d to the existence of sma~ ~ m e r poclke~s in the g e l s~e~ica~b ~nac~s~'i~e even to these v e ~ ~ s~ute t ~ e c u l e s ('forbidden ~ k ~ n ~ " o r "apparent n o i s e vent ~ a t e f . W~) (2]- A ~ o ~ e r ~ l ~ " ~ be that W~ represcn~ the amount of wa~er ~ is ~ghfly bound to the m a c ~ m o l e c u ~ r m a t ~ , , and for that reason not ~a~Iab|e as soh-ent [10,H]. In both cases w ~ = ~ - ~ . K~ ~a~ues> i are ~ e ~ by s~ute a ~ o r p t ~ a to the gel matrix [~.7]. In ~ t r a d ~ tion to ~ms assumption, hcm*e~er, it appeared ~ p ~ b~e in several studies to saturate the ~ binding sites in the gel malrL~: the e~u~o~ ~ m u e remained con~anL even at very h~gh solute ¢mmeni~rations [8]. Therefore, other imestigato~s suggeste~ a quite different ex#anation for the aberrant b e ~ " of solutes ha macromo~ecular g e l It ~zs ] p ~ that

the ge| m a u ~ has a pronounced influence on the s l n ~ w r e and function ~ internal water in the g e l thus changing its s ~ ¢ n t behavior | 3 J Z | 3 ] . If so, the elutkm of ~ ~ u t e frem a go! column ~ k ~ not only be de~nn~ncd by its m o ~ : c h r size, ~ t a~o by ~Ls partidon between ~he water ~n the cx~cm~, m o b ~ phase and the i n ~ r n ~ water of ~ e g e l Further, ff a macromo~cc~|ar ~arf~¢¢ affccLs the s l n ~ u r e and pr~pcn~cs of the vicina~ water ~¢~s. 1hes¢ phenomena should be regcvant to the s~ate and pr~per~es of ~ _ s m / c water, as pointed ou~ aga/n recent|y by W~g~ns and In the p~csent stodgcs the effect of c h a n ~ g ¢xpedm c n ~ con~itmns on the e l u t m hehavi~r of sm~! ~ u t e s from gel ¢ ~ u m n s was ~nves6g~tcd. It w~li be shown that the ¢xpcr~r~cnh~ re~ILs are in acco:dance with the nodon of an ~mcmal water phase, with a soh~nt h c h a v ~ that differs from the so~¢nt ~havu~r of bu~k water. M a t e r i a h aml Metheds S e g ~ e x G | 0 and G25 were o b ~ n e d from PharBdo-Ge| P6 ~om Bdo-Ra~ and PF.G (M~ 20000) f r ~ n Huka. 2"_.0 and ~ C - | ~ e ~ . . d s~utes were purchased from Amersham. In all experiments L5 × 90 cm, ~mpcrature-~onm ~ c d columns, loaded with p r e - ~ ' ~ n gel ~ ¢ r e used. g¢| was equilibrated with the e|~cnL buffered w~th IO mM phosphate buffer (pH 7.4) and c ~ m ~ n / n g 2 m M of the solute under study, befc~re the actual run. ~ n ~ , a 0 . | m~ sample, c o n , / n / r i g T 2 0 and the ~C-~d~He.d s d u t e , was loaded Ohm tim gc| and eludon ~ s sh~ed at a flow rate of 4 m ] c m - : h-~. Tim ~nt was confin~:~s~: monitored for r~atk~g-dv~. T h e Ko ~ u ¢ of the s ~ u t e was c a k ~ l ~ d fi'om Eqn. L Vo ~ s m e , ~ r e d ~]~h ~C-hhe|~ed inugn and ~ w/th T20. In some e x ~ r ~ n e n t s the resu|Ls are expressed in terms of "alq~m'ent non.soh~cnt water', according to W~ = V, - V~. A~ expcrimenLs ~ e r e run in I~r/p~a:a~, with an e x p e r ~ t a l error < 0_5% of the c a k ~ a ~ d Ko ~a|ues.

e000 b

c

~00~ ""

i!

: :

ii .i~

200¢

elution volume{roll Rg L l~ul~n curves on Scphadex G25 (30.2 g dry weight), a. la~L'~nulin¢ ( = VoW,b, |~4CIDMSO:c, 1"20 ( = V~);d. ~4C-thiourea.

results are shown in Fig. 1 and Table !. in all experinmnls the elution peaks were sharp and highly symmeb r/caL With sephadex G25 the W,~ for the five solutes de.creased in the sequence: galactose > DMSO > glycerd > gb'eine > urea, whereas with Bio-Gel P6 a quite different sequence was observed, viz: DMSO > g l ~ i n e > glycerol > galactose > urea. Th:. k-d and W~ values were not affected by the addhion of 250 mM K O to the eluent, nor by changing the pH in the range 4.0-8.5. With dimethyisuifoxide the effec~ of solute concentration was investigated. It appeared that K d and W~ values were independent of the salute concentration in the range 0.1 m M - 3 M. The effect o f t e m ~ r a m r e on the W,s a n d K a t'alues o f small solute molecules in Sel~adex GIO

To determine the possible effect of temperature on W,~ values, eludon of galactose and t-butanol from a S c p b a ~ x G I 0 column was monitored at varying tempcratores. As shown in Table I!, Wns decreased with increasing t e m p e r a t u r e both for galactose and t-butanoL Most striking is the fact, however, that Wn: (t-buhanoD is much higher than Wns(galactose) at 2°C, whereas at W C this is reversed, with Wn~(t-butanoi) < W~(galactose). At higher temperatures W,~(galactose) decreased further, whereas W~(t-butanol) became neg-

Results TABLE ! i n c o n ~ experiments it was ~ that the V~ values of the solutes was net affected by the elutmn rate. in the range 0.5-20 n~ cm -~ h -~. in aH subsequent capedments a flow rate of 4 n~ cm -~ h -~

K d and W~ ralz~esof some low-molecularweight solutes on Sephadex G25 and ~o-Gel P6 at 22~C

~as adopted,

s~ute

Apparent v . o ~ o h ~ ' ~ water f o r s o m e ~T~u~ solute nudecules in S e p h u d ~ G 2 5 a n d Bio-Ge2 P6

Galore DMSO

In a first serk's of experiments, the elu~ion behavior of five ~,.-mo]ecul~r weight solutes on S e p t 7 G25 and B~o-Ge| P6 columns was measured at 2 2 ~ . The

Gb~er~ G~ne Urea

W,~ is c,xpr~scd in m! H20/gram allyweight matr/x material ScpbadcxG25 W~ Kd 0.617 0°759 0_581 0.773 0.539 0.789 0.480 0.812 -0.170 1.066

B/o-Gel P6 Wn, 0.362 0.889 0.494 0.692 0.000

K~ 0.894 0.741 0.856 0.798 1.000

i / / _¢

_c - 0 2

QI 0

-Q2 ~

TABLE il

t h e effec~ o f ~ ¢ m p e r ~ u r c o n d~e elufion b e h a ~ m r o f several scqutes ~ a s im-~¢igated in m o r e ,telail. I n Fl~s. 2 and 3 p k ~ s o f tn K d v~. I / T a r e s V ~ . ~ f ~ w a g : - ~ s o l u t e s o n .%phadex G [ O c o l u m n s . T h e ~ exhort basically linear r e l a l k m s h l p ber~-een In K~ and I / T . tmwever. ~71h clear b r e a k i n g !~olms in t h e c~.~r~~ a~ temperatures~ l~sted in T a b l e i l l T h i ~ u r c a exhibited a K~ > I l - e r ~he entire l e m p e r a~ure r a n g e ~Flg- 2~. T h e r e f o r e . l h e effec~ o f ~he t h k m ~ a c i m c e n l v a l k m ~-as m e a s u r e d ~al HVCL I t app e a r e d tha~ t h e K~ ~ , s i n d ~ i x n d e m o f ~he th~.~rea c o n c e n t r a t k m in t h e r d n g e 0.1 r a M - 3 M. The effects o f dutien b e h a ~ Addition of n o u n c e d effect

on t~. Kd t ~

Temperature

GM~'I~

°C~

Of g ~ t m e , t - ~ t ~

~

~ ¢ L ~

~-b~a~

[0 -~)

0377 0-;65

0319 -O-013

TABLE ili

s~tcs c~ ~

urea and p o ~ d e ~ . ' ~ e g~ycd~ an ale o f small s d u t e rooks.des 6 M u r e a to t h e e l u a t e h a s a proo n t h e K,~ ~ l u e o f g a l a c t i c , t-~atanc~

TABLE |V T~e inF~cnc¢ of 6 M ~

T~u~e CC~

lire~ff6~gp~'ms { ~ 13.9

.~-~I:tm~l DMSO

16.6 !6A

~ i ~ r ~ on a ~

+ urea

5 |0

I).6t6 0.623

03~ 0__'~-,12

~} -t0 ~}

0.#r35 0.71)9 0.767

0552 0.~35 0.(X~2

0~16 0.68i 0.~7

1.!Y69 1.183

-13.~i .17.7

315

GlO cd~.n

Th~rea

t-t~t~'~

- ure~

c ~

Scsla~e Th~a

0.6?w'* 0.793 i.015

32.51 ~780 ~ 1~

~1 2.0~?~ I.TTO

1.I t4 133

] .4T2 133~

]~47 ]218

99 b

% RE~ a~

G]O ~ m

and thiourea over the temperature range 5-50~C {Table IVY. in the i~resence of urea the K~ wdues of thiourea and galactose were decreased, whereas the K~ of t-t~hanol ~as inc~ea~L Th~ effect of inc~ea~ng commntrathms of PEG in the eluent on |$~ of g a ~ o s e and thh]~rca on ~dcx GiO at I~P~[~ is shown in L~g. 4. In the absence of PEG W~(thiourea) is ncgat~¢. At increasing ¢oncentr~dons of of PEG W~, became less negafi~ {F~g. ,4b~. With g a ~ a c ~ the effects were ~ s i ~ e . In the absence of PEG W~. is p ~ i t i ~ , but its ~ l u ~ decreased ~ h increasing [PEG]- At PEG concentratkms ab~,e 1 ~ W~. ~ l y became negath~e (F~g. 4a). Dimassim The presented results confirm earlier obse~.ations on the anomalous behaviour of small so~u~ molecules in macromolecuku" ge| cotumns, with Kd values larger or smal~r allan unity. Theoretk~dly K j values smaller than unhy ndght be caused by decreased d ~ rates of sotutes in the stat[~naa~' ~ n t phase. As pointed out by Lanrent and KiUander, V~, and thus K d, ~ a h J be s~rongly dependent on the elution flow rate in that case [16~ As V~ values for all s ~ u ~ s u t f l ~ ! in the present studies did not exh~it any flow rate dependence, it can be concluded that diffusi~ rate limilation does not play a rote in the observed phenomena. Severed ebservat~ns ~ndicate that K~ values < 1 of small solute molecules can not be expla/ned in terms of •~cry narrow pores in the gel, hmccessib~c to the solute molecules. Very narrow pores will be more abundant in the highly crosslinked Sephadex G10, compared to the |ess crosslinked Sephadex G25. How~er,

Wn,(galactose) on Scphadex G2.5 columns is substantially higher than on Scphadex G I 0 columns (see Tables ! and i l l contradicting inaccessible pores as a major determinant of K~ values < ! for small solute molecules. Secondly, pore sizes in the chemically different Sephadex G25 and Bio-Gel P6 gels should be almost identical, considering their fractionation ranges of 10O0-.5~D Daltons for Scphadex G25 and 1O~]-(~00 Daltons for Bio-Gel P6. As shown in Table l, however, the W,,, on Scphadex G25 decreased (and thus the K,~ increa~d) in the sequence galactosc > DMSO > glycerol > glydne > urea, whereas on Bio-Gel P~6 the sequence DMSO > glyeine > glycerol > galactos¢ > urea was found. These quite different sequences indieate that there is no relationship between W~ and the molecular volume of the solute, as should be expected if K,j values < | are caused by the existence of small pores, inaccessible to small solute molecules. Further, in the presence of urea the K~(galactose) on Scphadex GiO is decreased, whereas the Kd(t-butanol) is incrY. If urea affects the size of small pores in the gel matrix, this would affect the K d values of different solutes in the same, and not in opposite directions. l~nall~; the obse~,ation that at 2°C W,~(galactosc) is h~ver than W~(t-butanoD, whereas at higher temperatures this is reversed (Table !1), can not be explained in terms of small, inaccessible pores. Also, the observed K d values > 1 can not be explained by simple adsorption of the solute to the gel matrix. Adsorption sites should become saturated at high sotute concentrations. Contrary to this expectation the elution profile of thiourea was independent of its concentration in the range 0.1 raM-3 M, with sharp, ~Tnmetrical e|ution peaks at all concentrations, Further, the effect of temperature on the K~ value of t-bulanol would he puzzling. In previous studies it was shown that K,t values > 1 of small solutes decreased ~ t h increasing temperature. This was explained by the well-known phenomenon of decreasing adsorption at higher temperatures [6,9]. With t-butanol, however, the temperature effect can not be explained along these lines. At low temperatures the K d of this solute is less than unity. At temperatures higher than 21°C, however, the K,s value exceeds unity, increasing with increasing temperature, making adsorp-'~on of t-butanol to the gel matrix an unlikely explanation. These considerations indicate that the aberrant bcha',nkmr of these small solutes should not be attributed to very small pores (K o < 1) or adsorption (K 0 > 1), but rather to anomalous solvent behaviour of internal gel water, as suggested previously by other invesdgatops [3.12,13]. Many studies have demonstrated that surfaces, either of solids or of macromolecules in solutkm, perturb water-water interactions, thus creating a layer of vicinal water with a structure and properties, quite distinct from those of bulk water [14,15,17-20].

This imp~es that s ~ u t e s ma.~"have a b~atkw a t e r / ~ c i n a t water distrr~mkm ceeff'm~ent d~ffering s i g n i f ~ ' ~ from unity [14,15] and ~h~ ~ k ~ fut~ + ~ n t for g~ • alues of small ~ m e s ~m gel ¢e~um,~ d ~ f e ~ g from un~'. More~'er. the p~esented r ~ k ~ can be reeducated eomplete~' ~i~h the ¢~¢¢p~ of a ~ ¢ ~ r ~ ~-ater phase L,~de the gels. F ' ; ~ . ~he h~:k of c ~ r r e l a t ~ between the ~ a r ~ u m e o f the solute and i~s K , value can be ex#ained e~sih; ~ith the ~'~nal ~ a t e r cc~ce~, in which the K~ ~Im: is determAned ~- ~he distribut~en mtk} of the s ~ u t e b e ~ e e n ~ I k arm ~k'ina~ water. This dism'~mtmn ratk} is rm~ expe~ed to be related to the m o l t e c u ~ ,,-~un'~. lint rather to the chemical nature of the s~ute. Furihero the different sequences of K ~ s ~ u t e } em g e h with equal pore s~ze bat different chem~al emmp~+it~ can be e ~ s ~ ex# a i n e d ~Table D. It is qu~te ~m.'e~-alge that t~e ~ vent prope~hrs ~ ~+kirml water at a Sephadex (dextran} gel surface will he different fn~m the+meof ~ ~ n a l water at a Bi~-Gei ~pe~.~'D|amid~:~ surfa~x:. The epgeMte effects of ure~ ~n the K~ ~Mues of differem ~ u t e s can also he rec~mdh=d ~dth th~ ~kinal water c~rmepL I~" breaking h y d r ~ a n b~mds urea will pe~ur~ the structure of ~ater. thus changing its .,~vent tm-+u,~ert~. Ctmce~-ab~'. the m@cnt p n ~ r ~ of bulk and +K-~n~ water v,-illbe affected to a quantitatN-eh- different c ~ e m . thus chart#rig the d i ~ n - b u t ~ ratm f~r ~ u t e s . Most important, the effec~ of PEG ~n the H~ and K~ values of ~ u t e s can be easi~- ex~a~ned ~.- the ~4c~nal water ~ . PEG w~ll affect the s ~ - e n t pmper~es o f the ~ d k water phase ~'~ the gel bed. as it ~-~'~1 create a v ~ n a l wa~er phase at its ~ surface. Fur~har. Wiggins and Van R.~ h~ve s h o ~ experkmentM~ that when a s~ub~e p ~ . m e r , l a ~ e r than the pm'es o f the gel is added to the suspenshm, the e q u ~ u m d~u-b butkm of ~ a t e r will be d ~ u r b e d and is s u b ~ - q ~ n ~ restored ~ ' ~ t e r expanding ~at of the p ~ e s . ~ea~kng behind ~-ater ~ t h a ~ e r derlsi~- than normal [14.15L This w-illchange the strue~ure and s~h-ent properties of ~he ~4cinal ~ t e r inside the gel p ~ n d ~ . These e~mbin~t effects apparently result ~n expelling t h ~ u r e a from the gel ~ t h im:reas~ng PEG c o r ~ e n t r a t ~ s . whereas ~ t h g~met~e the effect is ~ t e (Fig. 4}. Finally. the breaks in the cu~-es representing the relationship be~-een In K~ and I / T (F~gs. 2 and 3: Table l i d are in excellent agreement ~ith the ~ak~nM water concepL In many emensN~ studies it has been shown that the stracture of water changes abrupt~- at temperatures around 15 °. 30 ° and 45°C [17.1821]. The breaks obse~-ed in the present studies are very. near these temperatures (Tab[e HD. in living cells similar effects of structural (membrane} and macro~Mecular surfaces on the structure

and p m p e g m s o f ~k~h~ ~a~er L~er~ stmmid be expected, wi~h important ~ ~ ~ f~ the cell [14.t522]. F~r e ~ m # e ~ ~ and De Cger h ~ e slmwn t i ~ t a d ~ ~'ater ~ B ~ ~

tiered water ~ te~r~ ~ t h ~ r a ~ e s

~ffe~ the m ~ r ~ n [2~]- T h e r e f ~ e . ~ ~ e d

~ p~~ t M"

series of recem studies B~ggins e~ M. ~ d ~ e d ~hm in storm eases n m e ~ L m r geB m ~ be used ~ reLy-

results presemcd ~n ~his pager endegse ~his ~ m o f t~ghL

Rcqcmmees

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Me'~Ts. M-S_ Pere~rmev. V.D_ L ~

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155-167.

Acta lgO+ 17"2-176. 39-47. ~'tggins. P.M_ Van R.~m.R.T. and Orimm~ D.G.C. {lt~gD p ~ . ~. 60. 8-1~.