I"~ i l I OI I I O t . O ( l t "

Vol. 3, No. I, 196fi

B I O P O L Y M E R . S O L V E N T INTERACTIONS "["

JAKE 13ELLO

Delmrtn, ct, t oS Biophysics, Ro,~ivell /.'ark Memoria l Ins t i tu te , Bu~qalo, N e w York

In recent years the work of a number of in- . "' Nemeth3, vestigato~, especially ~eheraga and r- .,---

" "I':mford, ~'° and .Kauznvmn" :uid tlmir co- workers has established the hydrophobie bond as of prime iml)or~anee in the stabilization of ihe native eonformation.~ of proteins. .Also, hydmphobie denatur:Jnis are effective in dis- rupting deoxyribonucleic .~eid (DNA) struc- ture.'" '" I t might / )e [~t~et" t o use the term hy- drophobie segregation since the effect, arises r io t Ollt, o f a,n apolar-apol,.tr interaction that is intrinsically stronger than an apolar-water in- teraction. I t arises out of the unfavorable en- t r opy of transferrin~ "q)olar zroups to ~at,.r. It, ~a~ long exI~.o.ted that: rn t~t of the. apotar groups wotlld l m in file interior of a g]obuhtr protein; this ha~.~ been eonf inncd for "~ very., f,.~e " proteins by X - r a y diffraction, and Ires tmcn explained on tlmrmodyn:nnie grotmds. The recent work o~ " " ' ~ " ~eheraga an.d Nemethv ~-" ~'~ has a]so provided a stimulating concept of a moleeuh~r •basis, arising from a theory of water structure, for the thermod.x'namies of al)olar- water sy;stems. We also know that, I)NA hides its less polar portions from the solvent water, ahhoughli,~ this ease. tile ourine and pyrimidine bases have subst:antml chicle moments and are f a r more polar than the s ide c h a i n s o f such a m i n o acids as phenylalanine a n d leuein.e'

t tydmphobic effectshave become so p o p u h t r

in explaining the conformations of proteins

* Supported by Grant G M - 0 . ~ 6 fr~)m the In- stitute of Genon~l: Medical SCiences;: National In2 Stitutes of i Heal th , Grant.: NSF'GB.129 f r o m the National Scmnce roundation, and Grant .DRG#OI t'ronl t:hc . . . . . . . . . . .... " Damon Runyon Memonat :I: und ....

.I: rt~t=n,,ed a t the. ,<.,ccoml . . . . AnnUal :Meeting, ciety for Cryobiology, . . . . . . " "" ' . . . . . . . . . ~ ' Ia (hson, "~'~ScOilSm, A u g u s t ,1, ~ 1965i

that there tins l)een a tendency to overlook the previously fashionable hydrogen bonds a n d charge interaelions. Generally, these latter in- teractions are not sufficient to maintain lhe native tonform,ttion: but in many cases the hydrophobic effects are also insufficient. It. ap- pears quite likely tllttt,, the integrity of the over-all eonfonn.'ition depends on both apolar and polar interactions.

The stability of the interpeptide hydrogen bond in .tn a¢llleoH8 IEt(~([il.llll h a s bfff:ll the ob- ject of much discussion and some experimenta- t i on . .For the. dimerization of N-methylacet- nmide (NMA) in water, Klotz : lad-Franzen" fOilll{] that q.t; in~nite dilution ,A = 0. How- ever, Selmr:lga and Ndmethy TM suggest that in the NMA dilner the, re are metl.~yl-methyl inter- :letior|s with a posit.we AH, and lhat AH of the interamide hydrogen bond ..is actually some- what negative. Sc.helhn:ln" :rod Ndmethy and ~ 1 r*~ ., ..e ~e aga have suggested tha~ the interpeptide hydrogen bond has .a AH of about --1.5 to---2 keat per mole. At high NMA concentration, Klotz and Franzen ~ found a high degree of as- sociation. While much of. this rnust arise from the lower water content and nmrc favorable AS, it. is also possible t h a t . a l l is more favor-

4 al)le, than :it low water c o n t e n t . . F r a n k has pointed out tha% on forming a hydrogen bond between two water molecules, both:become more polar a n d can. form. stronger ,hydrogen bonds with other w,Lt.elr molecules. ,The sqme principle .applies to the forma,tion of peptide-water:hy- drogen bonds. I n dilute solution the:..-amide t.ro~a.bl) l . hsdrated as in the.. left s ide .o f

-4 ~ It " - " E q u a t m n 1, while in concentrated amide solu- t.i0il t h e situ'ttion is m o r e l i k e t h a t , o f , E q u a , t;ion 2:

H

. . . . . . . . . . tt:.,N .... n H ~ O --C~--O H--O ..... H ~ O + ' N i t : . . . . . . 0 . . . H---O ~--C~--~--O '.:.< " : ~r._.

• .. . H - - N - - +:.A.t~O - -C---~O H~O > + : N - H ~ ' , O H = , ' - ~ - -C~---O -- : ~ : ')" ~ l 1 t

27

(1)

(2)

28 j, BELLO

ci.cct of binding a In Equation 1, the ,v, second, or third, water to the one bonded to C = 0 is ~o m'tke ttm hydrogen of C = O ' - ' t t more pos~t)ve and to make lhe C - - 0 - - H

V * "1:* i l l - bond stronger than if only one water ~ (.~ .. voh'ed. , ,mnlarh. the N - - H - - ' 0 bond )s strengthened bee:rose the oxygen is more nega- tive than it would be if only one water were invoh:ed. Thus, interamide hydrogen bond- ing in Reaction 2 would be more exothermic than in Reaction 1. In a fairly compact protein of approximately native conforma.tion, the hy- dration of some of the peptide groups may re- semble tleaction 2 more than I. Thus, the ex- ten~ of hydrogen bonding and the eontribu*km

. ~ t ¢ " ° . 4 • ( of the hydrogen bonds ~.o the ~t,.~bfl~z:.tttm of the prot•ein may be grea.ler than one v:.:,~:~•!d ex-

' T L ~ , t J, peel from the AH of N-.~k dimerization. In different c.,t,..es • , ,a ,- the relative importance of

hydrophobic and polar effects will vary. .[n poly-L-alanine the hydroph.obic effect, seems to be especially strong; the a-helix, of this peI.)tide is stable to hot water, as a result of very, effec- tive hydrophobic intcn~ctions.'" '

As the temperature is lowered, hydrophabic e f- fee.t., t..~.(.ome weaker and hydrogen bonds more stable. These effects are similar to those produced by nonaqueous soh'ems. Generally.', such solvents provide a. more hosp~tagle environmem hydrophobic groups, and, as a result, of thew

,~-. :mA far fewer hydro- lower dielectric con,.tant gen'bonding groups, they stabilize hydrogen bonds. Thus, three effects m,t' y...- be expected: I) dena.tu,m, tion d u e t o disruption of hydropho- bic :regions; 2) slabilization of st:ructure as .'.t

cohols lower, the co.nlonnatm ~al Iransi(ion ' ~ " ' . 4 "1 ¢ ,¢ teml)er:ttltre (T,,) of l:.x,) me p, mcr(atic riboml-

c]easc. '+ Interestingly, this ene..t depends on the length of tlm longest +Ir:li~ht chain, not, on

) , the ,.otal number of Carbon :~toms. lYrhaps branched chain compounds would Im of v~lue in cryoprcservt-~tion, ]it, is of interest that Find- lay et ul, s have j'mmd that ritn3rmclease (l)Nase). is enzvmicallv active in mixtures of alcoim~ and water. Similar rt~ults have been observed for a number of other enzymes.

We h.-~ve recently .studied the enzymic ac- tivity of eryslalline P, Nase suspcnd~l in :,~

- C ¢ ,q) , , ¢1, mixture of sa~',: MPD (,:-meth3l--,,4-pentane- diol )

C H) 1

0 Ol:l H

and.25% water. ~ These crystals coatain al~.)ut o0/c liquid by vohm_m. There are diflieut-

a C t l'~ ) t lmr molecule ties in e,deul-ttir,~ the ' ' " '° v as lit i s not possible r.tt present to be sure how deeply i.tlto t h e crystal the actual region of re,- action penetrates . :0n the basis of reasonable assumptions as to the number of molecular layers involved and an exponential decay with distance in to t h e c~:stal, : i t appears that lhe RNase: molecul(~ iv the c~'s'tal am about :),s act ive a s in aque~,<ls solution. F o r a solution of RNase in the same medium (prepared by adding three parts of MPI) t o one part of

the . aeta~t y of ! I r a total aqueous: ){:Nase), , ' " ,'t mas,s: o f RNase i s equal, to tt~at of t h e same

result of. stabilization oI(.!~ydrogen mnd~, Or quantity of "~N ",~" inso lu t ion . The RNase in 3~ denamr"tt[ot~and form'ttion of a new hydro- solution of 75%, M P D is very highly aggre- gen,bonded confomaation. A case in point is g,i.ted,, tlmrefore, either . the RN.'4~: aggregates the behavmr o f lactoglobuhn m H~Ordmxane,. are very porous or . thesurface layers aresuper,: ,ks the dioxane concentration rises, the optical active.: MPD may be considered as a k i n d of rotation becomes more negative, indicating a isopmpvl: a!cohol-tert-butyl, a|cohol combi~mr disorganization of :nat ive structure; then the lion. I t w o u l d b e expected t l i a t ,5%• M]?D rotation •becomes more positive. Correspond- wotlH denature RNasc; : here aga in branching ingto these effects, the.b: ~,,,,flue, computed from cha in s ma.v : result ' i n Iessened denaturation. the optical rotary dislwrsion, curve, wh ich in - T h e fact that.RNase, in [~t.h. crysialline. . form dicates a:helie'd contcnti first shows : little and .in a g g r e g a t e s in:: solution i s enzvmically

" f f ch,m,.e, and then becomes muchmore negative, active d o ' > n o t necessarily mean that the con- Ttle observations are consistent with an unfold- formation :is native. A normative con[onna- ing of. I the:: PrOtein. with: disruption of t he :bu r ' t,ion might bd rcfolded bY a~bstrati.,, t ,o -anap- ted lwdr0phobicJregion, and formation of a proximately native a n d •,active form.. . :Such considerable ..amount of hydrogen-bonded a- an:effect.hasbeenobserved for RNasc denatured hdix. ~ Another case of. destabilization of :hy, b y 8~r.urea.;:.on addition ofsubs t ra te 0 r o f : a droptmbic-regions is t h e observation tim!t: al- polya~fi0n, refl~lding occurs and . enzymic a c

BIOPOLYM ER-SOLVENT INTERACTIONS 29

tivitv is ob~orve(t." In the case of RNase erys- t:tb+ in 75% MI'D, it is highly unlikely that tim molccuh-:s are ut~fohted to a random con- formation as it would i~'~ very difficult to pack r,"~n(lom!y tloppy chains imo a c~'st, dline lat- tic,_'. We h,~ve observed that.. ]tNase crystals do nm form unless 2 moles of phosphate or arsenate :Ire present I er mole of cn,~3 mc (other aniot~s may t~,, suitable); t h t ~ anionq may tighten and regularize the conformation, tin.r- mitring formation of erysudline lattice. There I s tim l)O:~sil:.,ility that lhe It.N,'~:.~ in the crys- t:d has disrupted hydrophobic regions but aver.all regularity as a resnlt of having more hydrogen,bonded helic:~l regions. This would n~quire that the binding of a molecule of sub- stt",tte reverses the effect of the solvent. On the whole, ii is not. unreasonalAe to consider Ihat l lNase in 75% MPD is of substantially native con fo n~l,'.l I ion.

The. use of phospha.te to obtain t/.Ntise eD's- tais snggi~ls ih,,lt some multiply charged anions be added to eryoproteetive solvenis. Of course, such ion~ are already present in cells, but per- Imps more should be added. The effeei..~of the :~nions al)Pears to be related to tile observa- tions that tilt; conformation of a,. protein is protected a~ainst proteolysis as a result ()f binding small,.moleeules of various sort, o;. tiffs has been explained as resulting from the rela- tive st-ff)iliTzttion of .one or a few conforma- tions out of the multiplicity of conformations that a protein may pa~s through os-a result of thennat motio,~s)' Perhaps some cryoprotee- tire solvenis liave such an effect.

A diff(,..rent ' " ' concept of denaturation by o r - ganic solvents has been advanced b y Sinanogh., et aLY and appl ied by- them to DNA. ~.~en- tially, this involvest.he calet~!~.,tion of the work required to generate the "~dditionaI volume of c.v~vitv .required t o aeeoinniodaie the..[btdkier denattm:d fom~ ' as. compared, to :.tim ~ :native

can occur. For ex-'m~ple, while 50% dioxane unfi)lds fl-lactoglobulin, 90% ethylene glycol is required. "~ Also, Sage and Singer'" found that the 'dmormal ~yrosincs of RNase are nonnaI- ized in pure glycol. At 75% glycol, we have found that the normalization is about two- thirds. In some of these solvents some proteins may t:~:: more sts4ble t hfm in water. For example, the T., of the collagen helix is rtfised in glycol or glycerol. The rate of formation of the helix is re- d,Iced, l:mt th is is probably .~ result of the h:l- cre~,~sed viscosity, The cause of the increased T~, may be: l) stabilization of hydrogen bonds; 2) formation of stable intramolecular glycol or glycerol bridgt.'s'; or 3) aggregation. Hydrogen bonds may be important as collagen is a protein poor in apolar side chains, although one. can- not he sure how to classify a side chain as that of lysine which h a s a polar end but an apolar middle ~,.ction. ,~ven ty - f ive per cent glycerol raises the T,., of RNase, a more typical globular protein, b u t one lhat lms also a pau- city of nonpolar groups (about 25% of leucine, isolcucine, phenylalanine, tyrosine, and va- line. I t is possible t h a t tile: t ransi t ion.being measured is not rmtive ~ .denat;ured but: native

regul,tr, non-native -+ denatured. The effects of solvents will vary from, pro-

re in to protein: a n d f r o m membrane to:. mem- brane,, T h e ~ effects will va ry .dur ing H..O solvent and: solvent. ~;~:,,H,O .exchanges in warm ~..eold and cold --, warm transitions.. In some solvents, enzymes may be inhibited; in other solvents, certain enzymes may .be ac~.ive, and tl~e substrates very accessible, leading)to ir- reversible:stnmtural deter iorat ion. In ~ some :sol- yen ts, denaturaemn of ~. some _biopolsmaers :.may. b e irreversible; :: in.:-others, h ighly reversible. Herskovit .¢ . . f0und-])NA denaturation by al- eohoks to"be more reversibleS.that-denaturation:, by hol:.water.

"Ill addition t o the "'direct!'. effects, of. solvents form: The resul.Ls obtained are m agreement on biopoLmmrs, there: are al .file

" t - "

with the • • , • , ." ~" Small . molecular components:: as , experimental . /o rder :of::effectL mle,.~ Of a var ie ty ofsolvent:s such asaicoh01s, glycol, sattS[[iiPids; steroids,/andothers/~/For:examplei- glycerol, and formatnide. "l, :"cimnge: of sol veni~" may. cause.~ sinall mblecules

It. iswell known thatpolytE,:droxy compounds to .: associate ..."or diasocialte::- from~::!bi0polvmer structures, resvl t ing .ill ei ther, s~biliz;afion o r are good protective agents.: ThiS- is predicted

by Sinanoglu's ca.leuLa, ti0ns, and. has been. fOund for t h e denaturat ion-[of protein's and lmcleic ilCids[ Tliese solvcn~:s -~ :do not: den/iture proteins as .readily. as .do nonhydroxylie solvenis, but at sufficientiyliigti..c0neenixafion denaturation

laboratory,: :iwitll: interesting r~Uits.:,For:: e,x,-am- ple;-: the:, meI fifig (p0int: of-:)a );: 3%:,: gela!ih):gel:!.: is 'affected b~,

30 a BLLI,O

1 ABLE 1

M I.~I ,TING I ) O I N T S O F ( , I g I . , A T I N ( t E I , S

W a t e r i 7 ~' '1 ) - r., ! S.:,~ G ycol i ~5 ~a: G l y c e r o l ' I

Salt } ~

',~t'

(N EI~) =SO~ N a O A e TMA C1 Control NaCI

Melting { point ]

!

I 30,6 f + 2 . 4 30.0 l 28,8 } + 0 . 6 2 s 2 } 0.0 26.4 ! - 1 . 8

~ 25.2 1 - -3 .0 ...4b ! -3.6

, i - - 3 . 6

1 9 . 0 I . - 9 . 2 - 1 , t . 8

1

NH,~CI Urea LiCl GCI$ N a S C N

I M e l t i n g i r, oint i ~' i A , ,

1 I i [ 1 2 ' ) ~ - - 3 . 2 I i "" { . . . . ,~ 3 3 . 6 t - - 1 . 8

30 4 0.0 I . ,

i 2 7 . 2 - - 8 . 2 . 27 .4 ~ - - 8 . 0 i aa.4 { - 2 . 0

2 7 . , t I .~ ~ - 8 . 0 } 31.0 ' ! - 4 . 4

1 3 . 0 ! - 2 " 2 . 4 ; r

I M e l t i n g { & a p o i n t ' ? ) ! *

I ' }, * q ", , 4 0 . , t f - 0 . , : - . . 8 " . 4 , 4 i + 6 . ' t

- - 1 . 8 +10 .2 0,0 +12 .6

") + 6 o

+13.8 + 8 . h

+1.5

+ 2 . 2 t 36,4 + . ) . s i 3~.0 t + 7 , 2 1 4 0 . 8 ! +0.~,.. 1 32.6 ! + 2 " I '~ "g' i

i • g : 9 + 8 , 8 3,..4 ; - . , . , t b2:8 ~ "' - ~ 3 3 . ' 2 : . : -7 .~ i

+ 1 . 2 i 33.8 { --7.0 - - 0 . . t I

*:at is the change m melt ing point compared with the control in the same solvent . I" A~. is the c.hange in melt ing point compared with tha t of the sqme addi t ive in I:1=O. {: Guanid ium chloride.

gelatin gel is closely related to the molecular conformational transition temperature.) Some of our results are shown in Table 1. I t is to be noted that, whereas in H~.O ammonium sulfate and sodium acetate raise the melting point above that of the control, in the glycol and glycerol they lower the melting point below that of the control. Also while NaCI in H=O has onh' a small effect on 5/',, in glycol and glyeerol it is as effective as LiCi. Indeed all the additions of the table are better denatur- ants in glycol and glycerol than in H=O except urea and guanidium chloride. Possibly other additives can be found which may-protect in eryoproteetive soK'ents even though they are denaturants i n water. Some of the normal mnall molecules of cells m a y ,become denatur- ants in cryoprotective sa~Ivents if they become concentrated in certain regions.

REFERI3;x CES

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2. 13ixon, M . , Seheraga, i:l. A., and Lifson, S. El- feel; of hydrophoMc bonding on stability of poly-L-alanine helixes in water. Biopolymers, 1" 4 1 9 - 4 2 9 , 1 9 6 3 .

3. Findlay, D., Mathias, A. P., and Rabin, B. R. The active site and meellani...~ of action of bovine pancreatic ribonuelease. Bioehem. J:, 85: 134-139, 1962.

4. Frank, l:I. A. Covalency in the hydrogen bond and the properties of water and ice. Proe. a-Roy. Soe. [Biol.], 2~7A : 481-492, 1958.

5. Gratzer, W. B.. and DoW, P. A conformation

exanfination of poly-t.-:,hmmo and pOly-D,L- alanine in aqueous solution. J. Amer. Ch(:m. Sot., ,..o. 1193-1197, 1963.

6. He.rskovits, T. T. Non-aqueous solu(ions of .lr)~..A: }actors determining the staMlitv Of the helical config-uration in solulion. -~,reh.

• • " 9,,. 47.t-484, 1962. Bmdlem, Biophys., "" 7. Herskovits, T. T. Non-aqueous sol.utions of

D N A " Denaturat ion by urea and 1~,. methyl derivatives. Bioel'emistry, 2: 3,a5-:~0, 1963.

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9, Klotz, 1. M.. and Franzen, J. S. I t yd rogen bonds l~etween model peptMe groups in so- lution. J. A mer. Chem. " • """ ' " " " ,:.~_m., b.".i 3.t6,.-3,t66, 1962.

10. Levine, L., Gordon, J. A., and Jeneks. W. P. The rehitionship of the structure o:[ t!m ef- fe~;tivene~ of denaturing agents of deoxyri- bonucleic acidl 13k)chemistry, 2: I6,%175, I(,)63.

1I. Markus, G. Protein-substmte conform,,~tion and prot(.ols.-,ls. Pr(~. Nat . Aead. r am. . .U .S .A . , 5d : 253-.258, 1965.

,y,r ~.~ ¢ ,) 12. Ne'ni-tt. ix',. (.I., and Scheraga; I:I. ..~." l he stnlc-

ture of waiter and hydrophobic t:mnding in proteins. I. A model for tim thermodynamic. properties of liquid water. J. Chem. Phys,, ,%. 3~:kg2-3,I00, 1962.

13. N(.'met w. (.,., a n d $chcraga, H. A. The struc- ture of water, and hvdropho})ic. . . . . .: [~.mdln. . g in proteins. 1II. The thermodynamic properties of hydrophoMe bonds in proteins. J, Phys. Ci em 6!I: 1773--1789, 1962.

1,1. Ndmethy, G , Steinberg, I. Z., and Scheraga, ,H. A. Influence of water structure and o f hy- dropl-mbie interactions on the strength of side chain I{[-bonds in proteins. Biopolymers,

. ,I.R-(;.0 I . . . . . . . . . 1963. 15. S:~e, H. J.; and ,.roger, S J~ The properties of

]]IOJ)OI,Y'M|.R-SOL~ ]..,N I IN FLRAC I ION,_ 31

bovine pancroatie rilmntwloasc in ethylene glycol ~ohttion. |liochemistry, 1: 305-317, 196~.

16. Schellmnn, J. A. Th,.,rmodynamics of urea so- lutions :rod the ]mat of forrnation of the l)cp- t ido hydrogon l~ond. C. R. Lab. Carlsberg [(:'.trim.}, 29 : 223-229, 1955.

17. Schrier, E. E., and Scheraga, ]l. A. Tim effcct of aqueous alcohol solutions on tim thermal transition of ribomtcloase, l~,iochitn. Biophys. Acta. 6.~: 406.-.lfkg, HN2.

IS. Se°]a, 31., Anfin~:m, C. B., ~tnd ]ilarrington, W. F. Ttw :corrvlation of ribont~cleaso activity with sll,.,cific asriects of tertiary structure. Bio,rhim. P, iol:,hys. Ac~a, 26: 502-512, 1957.

19. Sin;mol~lu. O., AbdulmJr, S., and Kestner, N. R.

In, 2 ~(, ." , Llc .tromc asl)ects of biochemistry, B. Pulhnan, ed. Academic Pre~s, 'Inc., New York, 1961.

20. Tanford, C. C0ntribution of hydrophobio in- teractions to ihe stability' of the globular conform:d ion of p.roieins. J. Amer. Chem. So(:., 8,~ : 42,10-,1247, 1962.

E.. De, 21. Tanford, C., But'klev,. C. I~..K.," and Lively, F,. P. EffecL of ethylene glycol on the eonfornmiion of a-glohulin and ,,3-lactoglobu. lini J. Biol,.(.,lt(!m.,z3¢ 1168--11~1, 1962.

,w a . d , "~¢ • 99 "l,mtord, C., I)e, l'. K.. and Taggart, "V. 0. The role 0f the a-holix in the structure of protein.s. C)t)tic.al rotqtory dispez,'sion of t3- lactoglobulin. J. A i r i e r , Cho.lla. S o t . , 82: 6 0 2 8 -

F-g33'|, 1960.