Nature of urea effect on anion binding by macromolecules

.\RC‘HIYES

OF

Nature

BIOCHEMISTRY

of Urea

BSD

BIOPHYSICS

Effects

123, 551~.?.57 (I%%)

on Anion

Binding

by Macromolecules

The binding of methyl orange by bovine serum albumin is srlppressed I,?; ,~a. HOW ever, the inhibition is largely reversible if llrea is removed either by dial;vsis (fast or gradient) or 1)~ dillrtion. Binding by a synthet.ic polymer, polgvinqlpyrrolidonr, which is unordered and swollen in water, is also suppressed by IUW,. Thrse observations indicate that llrea effects are mediated largely through its ac.tir)n on the solvent.

It, has been Imow~ for many years that thcl addition of urea to aqueous solutions abolishes the binding of small anions by prot,ein molecules such as serum albumin (1). However, t,he nature of this perturbation is still not, clear. 14’~ :i long period (2) it was assumed generally that urea disrupts the native conformation of the protein by forming hydrogen bonds with the peptide groups, and this viewpoint ha:: been recentI\- supported by new solubilixation experiments (3). A1nalternative, more recent explanation has been that urea \\-e&ens hydrophobic bonding by competitive clathrate formation (4) around npolnr side chains. It has also been proposed that the influence of urea ma\- reside in its efl’crt, on the solvent, for example, in its intwfercnce with ordered water st,ructures surrounding apolar residues (5, 6). To investigate furt,her the mechanism of urea action ux have carried out experiments on the reversibilit>. of this interact,ion in nnion~albumin binding and have also examined urc:~ effects on binding b)- a synthet ic macromolecule, polgvinylpyrrolidone. 1 l’rrserrt address: Biological Institute. Tohoku l-rriversity, St=rrdai, Japan. 2 This investigation \vas srlppcbrted irl part hy a grant ((iI3-lG10) from the Xatiollal Science l~c,lultlalioll.

.l/u(eriu/s. Crystallized l)oville serum alhrrmin was pnrchascd from t’elltes, Inc. The moistrlre ccmtelll was determilled t)y drying a sample ill an Al~drrl~altlr~~ pistol in vaclu) over P&r, at 110”. Initial protein concentral iotbs were driermined by ~vcighil~g. 111the course of tlcliaturaI ioll-renaturnt iou experiments, changes ill collcentratioll \vere mrastn-ed hy reading the ahsorhallce at 280 mp. In phosphatr buffer, 111-Ifi.!), El:.‘,j, \vas fo\md 10 be 6.88. Phosphate bluffer of O.l:i ionic strength nut1 pII 6.9 was prepared from O.OY-l!) 11 Xa~lIPC)4~ ill& and 0.0277 JZ KIlYPO,. A reagent-grade i,\lerrk au1 Co.) methyl or:tllge was recrystallized from water. The molar estillction coeflicielll at. 464 111~was 26,900 1t-l en-l. I-ren (Baker Analyzed Reagent) was dissolved it1 u-ater at, 50” to form a saturated solution, and the soltltioll was filtered alld ru’ea was cr~st~allizctl irr the prcsellce of %O% methat~ol. These crystals were dried ill a V~CII\II~ over l’.lO:. Fresh IIre:t solut iolls in 11111Tcrwere prrpared for each eslleri-meut. 1’ol~vi~~~ll~~rrolitlo~~e Ii30 was oht ainetl from C;cneral ilnililre nut1 Film Corp. According to the rnanufact~lrer, this type has :a mcAeclllar weight of Rl)ollt 40,000. I’olJ-=a-r,-lysiile hy(lrc~bromidc was prlrrhasecl from Pilot Chemicals 111r. Lot L-M had a weightaverage m&xldar weight, of W,OOO; lot L-1i6, au A1?T,, of 95,000. Hromitle ion was replaced by acetate as follows. Thrrr gm of polylysillc hydrohromitle w-as tlissolved in 150 1111of acetate buffer ~11 6.57, ionic strength 0.1. This solrltioll was dial!-zed ngailrst 2 liters of I)llffcr for a periotl of 2 days \vit,h

5.51

(i:jz

KLOTZ

AND

freqlt”nt changes. Buffer was t.hell replaced by distilled water to remove excess acetate, and further dialysis carried out for 2 days. The solution was filtered and the polypept,ide was recovered bJ lyophilizatiou. After this procedure the product no longer had an aromatic odor, and it gave no test (with silver ion) for bromide ion. All salts and sllcrose were reageut-grade materials. Equilibrium tliu(/~si.s. The extent of binding of methyl orange was measltred by equilibrium dialysis according to the specifications of Illlghes and Klotz (7). Ten ml of 0.2y0 serum albumin or of polymer inside a dialysis bag was equilibrated for abollt 20 hours with 10 ml of methyl orange solution. Control trtbes contained only bktffer inside the dialysis bag. Eqt~ilibrillm methyl orange cog centrations were determined from absorbance measurements at 464 mH. Gradient ezperijtrents. Some derlatllr:ttiotr-renaturation cycles were carried out by gradual changes of solvent composit,ion with a gradient device. A dialysis bag was fastened t,o a glass tube (1.5 X 18 cm) projecting partly illto it. Two 3-mm polyethylene tllbes were inserted throrlgh t,he glass tube into the bag, and one of t,hese served as inlet

FIG. 1. Binding of methyl orange by bovine serum albumin in phosphat,e buffer, pH 6.9 at 25”. 0, Xative albllmin; q , albumin ([(Y]D = -58”) dissolved in 8.3 M urea ([a]~ = -loo”), allowed to stand 72 hours and recovered after dialysis ([a]~ = -58”) to remove tlrea; A, albumin ([a]~ = -58”) dissolved in 8.3 M Ilrea ([a]~ = -lOO”), allowed to stand for 24 hours, and recovered after gradient for 50 hours to remove dialysis ([a]~ = -58') urea; V, albumin ([a]~ = -58’) dissolved ill 8.0 M urea ([LY]D= -94”), allowed to stand for 24 hours, and recovered after gradient dialysis to remove urea with solution containing 1 X 10-s M methyl orange drkng dialysis.

SIIIKAMA TABLE BINDING

I

CONSTANTS FOR METHYL OR.WGI~ PHOSPHATE BUFFER, pH 6.9-7.2, 25”

IN

hfacromolecule

Native

bovine

serum albumin

Renatured bovine serum albumin” Transferred from 8.3 urea to Transferred from B..i urea to Transferred from B..i urea to Transferred from 8.5 urea to Polyvinylpyrrolidone

Y 0

3.9

0.2

3 ..i

1

2.4

2

1.5

0

2.7h 0.28*

M M M

6

a The specific rotation [a]~ returned to -58” (t,hat, of the native protein) when serum albumin was returned from 8.5 M to zero M urea, no matter whether urea was removed by gradient dialysis, by dialysis against buffer, or by direct dilut,ion with buffer. e Calrldated from T values computed for 1Oj gm of macromolecule.

for the ,lrea solrltion coming from a gradient mixer and the other provided an exit. When 10 ml of urea solrltion (from 0 to 8.5 M, iu phosphate buffer) had initially filled the bag, it was inserted into a 3 X 15.cm test tube contairling 20 ml of 4% bovine serllm alblmiin in phosphate buffer. The protein sollltioll was stirred constantly to facilit,ate eqrlilibrium with the gradient solution inside the bag. The llrea gradient, was chauged slowly (O.‘t.S-0.5 M per holu-). The urea concentratiou was measllred by followiug t,he refractive index of the orltflowiug solrltion. This same device was used tr, remove [Irea gradually from a serum albumin solution. In this case the urea gradieut was from 8.5 to 0 Y. f
(.P,EA

EFFECTS

ON ANIOPU’ BINl)JNG

Kewrsibilii~

UREA (Ml

LOG (Nhee FIG. 3. Effect of 1 M urea 011 binding of methyl co-:tnge by bovine serum alhllmin in phosphate b~tffer pH 6.0 at 25”.

oj

uwn

c$ect

on

cdhzc7mn

I’igure 1 and Table I compare the extentsof binding of methyl orange bJ* nat,ive bovine serum albumin and by albumin that, had been exposed to S M ure:~ and freed from urea by dialysis. The graphical representation presents ‘T, the moles of bound anion per mole total prot#ein (S) versus the logurithm of the concentration of free, unbound anion. lcigure 1 includes only the data for r <, 1. It is clear that exposure of nlbuniin to S 11 urea at 25” for periods up to 72 hours does not, abolish binding ability if the urea is removed. It does not matter whether urea is removed by gradient dialysis, dialysis against vvatcr, or direct. dilution. At, low corlccnt,rat’ions of methyl orange, binding by regenerated protein is almost. as strong :I!: t,hc original native substance. At higher concentrations of free met,hyl orange, particularly in the range 1 < I‘ < 10, t#he binding curves deviate more strongly. For quantitat,ive comparison, values of kl, the first, binding constant, have been obtained by suitable extrapolation procedures (S), with primary emphasis on the high concentration experiments, and these are listed in Table I At zero fina urea concent,ration, k, changed from 6.1 X 1OAfor the nat,ive protein to 3.9 X lo4 for renatured albumin. In terms of free energies t,his amounts to a difference in binding affinity of about 300 Cal/mole. At, this point one might n-ondcr whether in t,he presence of urea t,here really was a strong effect on the anion-albumin interaction. However, t,hc earlier observations (1) Dinclinq.

FIc;. 2. V:Lrnticlrt it1 absorlxtrtre ctf methyl otxlrge with tIrea wnceutrntion, in presence of hoville wr,ml ;ilhllrnill (BPA, and with buffer alone. SIeth\-I ,,r:*l*ge, 1.2 X 10M5 31, WA, 0.3%; pH Ci.!)-7.4; 2.5”.

.-I.-l3

BY ~IACI:OP\lOI~~~:I~I~l’S

554

KLOTZ

AND SIIIKAMA

have been confirmed by spectroscopic shifts and by equilibrium dialysis. The spectrum of bound met)hyl orange is shift,ed to lower wavelengths, and hence the absorbance at -170 rnp, a \vavelength neal the peak for unbound dye, is lowered in t,he dye-albumin complex. When bound dye is dissociated, therefore, the absorbance at 470 rnp increases (1). Figure 2 show that the spectrum of d!-e ll-it’h albumin a,pproaches that of dye alone as urea is added. Thus there is increasing dissociation of dye from its albumin complex with incrensinp. additiotls of urea. It is clear that even 1 11urea has 2~dissociating effect’ and that interference Jvith binding of methyl orange is total I\-hen the urea concentration reaches 6 AI. These spectroscopic observations \vere confirmed at one urea concentration by equilibrium dialysis experiments. AN Fig. 3 illustrates, binding of methyl orange by serum albumin is definitely lowered by 1 M urea. Quantitatively, 1~~is reduced from A.1 X IO4 in the absence of urea to 3.7 X 10” in 1 31solution (Table I). That, urea denatures our samples of bovine serum albumin is also evident from optical rotation and viscosity measurements (Ta-

BSA

d / 0 i i/J> PVP

0

0

0

TABLE THERMOIIYPJ\M~C METHYL T.\'I'E

III

P.IRAMETERS ORASGE l~UFFER

a Calculated macromolec~de.

(M =

-1

-5.0

-4.5

LOG (AjFREE

Frc. 4. Comparison of hilrding affinities of bovine serum albumin (MA), polpvinylpyrrolidolle (PVI’), and polylysilre (POLY-LYS) for methyl oralrge (-4) iu acetate brdl’er, pH 5.6, 0.1 ionir strength, zrtld 25O.

RINDING

OF

ACE-

0.1). PH 5.67.25”

from r v:tllles compitte.1

for 101 g of

ble II). The specific rotation, [a]“, and t,he int,rinsic viscositJy, [T], change in the expected direction and by a11 amount ch:lr&eristic of this protein I\-hen it, is denatured. Thus it is apparent that urea produces well-recognized effects on serum albumin which are accompanied by a loss in the ability of this protein to bind anions. Whatever the nature of these effects, when urea is removed, the binding ability of the protein is largely regained. Furthermore, if urea is added to this renatured prot>ein the frsctional decrease in binding of methyl orange (Table I) is approximat,ely the same as for a corresponding experiment wit,h the native protein. I~fffvt oJ uwa. on binding by polfpnylpyi*~~olidone. A protein is a polymer of some twetlty different, t!-pes of residues, all of which, in addition to the peptide linkage and the solvent, could be intjeracting with urea. It therefore seemed wort,hn-hile to t,ry to simplify the question by examining urea effects OII binding by a simple homopolymer, with one type of residue. Some experiments were carried out first \vith polylgsine, :L homopolypeptide which is water-soluble and which, because of its large concentration of cationic groups, might be expected to bind methyl orange avidly. Surprisingly, binding by this polypcptide was n-cak (E’ig. 1). Consequently it did not seem promising for examining the effect of urea. It has been known for mmy years that polyvinylpyrrolidone(1) forms complexes with :I variety of small molecules (g-14).

POOLY-LYS

-5.5

FOR

ISY M.~cRo~I~L~~~-I,Es;

II 1

I,C __

CH, n

6 M UREA

This n~:~croi~~olecule is particularly intcbrestillg ilr that it can form no intrnmacrw molecular h~drogcn bonds, since it has no S---H do~wr group. Severtheless, it is WQ n-ater-soluble and parallels serum nlbuniin in m:tn~- respects in its interactions n-ith anionic and neut,ral organic molecu1r.c. A quantitative comparison \vas first made, thcreforc, I lt’ the aflinit’ies of polyvinylpyrrolidorrc ai~tl serum albumin for our standart nniotl, nwthyl orange (Fig. 4). The s>-nthetic pc~l~mer binds this anion to about one-third the cstent found in serum :dhumin. The nature of the interaction is similar in man? respects. l’c)l\-vin!-lp\-rrolidone, like strum albumin, she\\-s dccrensing affinity for small molcrule5 of different charge (1.5) in the order, aiiioils > neutral moleclllcs > cations. Furthernlorc, the thermodynamic par:unctcrs of binding are similar. In both ruhes the withalp)- of binding is :l snlall cxothcrmic cluantity and the elltlUJ)y is a substantial posit,ivc ilumber (‘rahlc III). Thus for both types ()f’ ii~:~croiiioleculc, formation of the anion complex is to a large extent an entropydriven process. L\S \vns JN3illted out lxm,I ~-cam ago (16. li), the positive entropy for Ihc formation of a complex from separate species is surprising and strongly indicates th:at the solvent plays a major role in this association renction of the two solutes. The effect of urea on binding 1,~. polyviri!-lp?-rrolidorle (Fig. 5) is vcq- marled. Dialysis espcriments in 6 11 urea slion- ;I strikil~g drop in binding ability in this solvent (Table I). In this regard, then, also the eyn-

-5.5

-5.0 LOG (A),,,,

thet,ic polymer behaves similnrl\albumin.

-4.5

to serum

From t)he observations described here it is apparcnt~ t,hat the suppressive effect of urea on binding of small molecules is not limited to strum albumin. It is also exhibited in a i~onpeptkk pOlynm. he wii conclude unequivocally, therefore, that, the mechanism of urea action is not limit,ed to disruption of S-H. +.O=C bonds or t,o breaking of hydrogen l~oiids betSn-een met,liyl orange and macromolecule, for these do not exist in p~~l~vin~lp~rrolidone. A similar conclusion has bren rtached previously from observations of the effect) of urea on the acid-base behavior of organic molecules covalcntly conjugated to proteins and I-,olyvin~lp~rrc~liclorle (13, IS). From a number of other sources it is also apparent that urea can affect the behavior of a variety of different synthetic mncw molecules, as we11 as biological ones, in aqueous solution. Recently, Hnmmes and Schimmel (19) have shown that, urea perturbs the ultrasonic att’enuation in aqueous solutions ot polyet,hylene glycol. l~urt~lrcrmorc. Bnrone et al. (30) have found that urea cluenches the affinity of (non-ionized) poly-

5.X

KLOTZ

AND

SHIKAMA

methyacrylic acid for hydrocarbons, as demonstrated in solubilization experiment,s in aqueous solution. Viscosity measurements for polymer in G p. 0.3 al urea have been made for polyethylene glycol (19) and for polyvinylpyrrolidonc (21). 0.2 In the former case there is a small change 0.1 ( < 10 ‘;I; in intrinsic viscosity) compared with n water; in the latter case, none within experi400 450 500 550 mental error. Thus urea produces no change A (mp) in the swollen unordered conformnt~ion of FIG. 8. Absorption spect,ra of methyl orange polyvinylpyrrolidone; yet it, has a striking bound to polylysine at. various valtles of T, the quenching effect on the binding affinity of moles of bound dye per lo5 pm of polymer. Acetate this macromolecule. br8’er, pII 5.G, 25”. 0.5 F-r--,]

400

450

500

559

A (mp)

FIG. 9. Spect,rum 2-p)-rrolidone :

of methyl

orallye

in (liquid)

A (mp) 6. Absorption spectra of methyl orange boluld to bovine serrm~ albumin at various values of T, the moles of bolnld dye per mole protein. Spectra recorded as difference spectrum between inner and otrt,er solutions of dialysis experiment in phosphate buffer, pH G.9, 25’. FIG.

400

500

450

550

A (mp) FIG. 7. Absorption specka of methvl orange bound to polyvinylpyrrolidone at various values of T, the moles of baud dye per lo5 gm of polymer. Same conditions as in Fig. 6.

It is also of interest to note that urea disrupts binding of methyl orange both to serum albumin and to polyvinylpyrrolidone even though spectra indicate t,hat the dye is in a different type of environment in each complex. Spectra of bound dye n-em obtained by measuring the absorbance of a solut,ion of the macromolecule-dye complex take11 from within t’he dialysis bag versus that, of solut,ion outside the bag. Since at equilibrium free dye is at the same concentration inside and outside the bag, the difference spectrum is that of bounddye.” Methyl a Corrections were also made for a small contributioll due to light scattering by the protein.

--c .).)I

orange tx ~md to serunl albumin shows an :thsorption maximum (Fig. 6) near 43.5 mF, but the complex with polyvinylpyrrolidone has a peak at, 470 rnp (Fig. 7) as does that’ n-it,h polylysine (Fig. 8). The free dye in bull; \vatrr ha:: :I peak near 4G5 rnp, but dye dissolved in pure liquid monomer, vinylpyrrolidone, ahcn~s a maximum at 430 mg (Fig. 9) and in ethanol or benzene (containing a trace of :~lcohol) at 420 rnK. Shifts to n-avelengths near 420-435 mk presumably reflect an :~pol:~r environment ; peaks near 4B.5 nifi, :III aqueous environment. Thus dye bound to I)olvvin?-1DSrrolidone is in a much more waterlike environment than that bound to wcrum :dhumin. Of course the polymer is a much mow s\vollen, open macromolecule than i:: the protein, as is indicated by the respective intrinsic viscosities, 22 versus 3.S (gm,/ ml)-‘.

Since urea has similar effects on aqueous solutions of synthetic and hiopolymcrs of very different structure and conformation, it. seems ut~liltely that it exerk ik pert,urbation by direct combination with these differcllt macromolecules. The very fact, that large concentrations of urea are necessaq for a denaturing action indicates that its effect occurs only when the solvent it,sclf has been siguificantjly changed, i.c., that the effect is :~n nlloplastic (6) one. If the action of urea is manifested through the solvent, then it, becomes apparent that macromolewlcs of \-err- diff went, structure and conformation can he affected similarly since they arc all being examined in a common aquec)u:: solvent.

F. 1. KLOTZ. I. &I., TM\\ I-MI, II., \NI) W\LKEI:. M., J. Am. C'hetn. sot. 70, 29% (1948). 2. ~~RSKY, .i. I?., .\NI) F’.\ITLING, L.. I’roc. .\;ct/. .Acctd. Sci. C'.S. 22, 439 (19Xj. 3 (:o~t~x)s, J. A., ASI) .Jmrr
I. 11.. Science 128, 815 (1958).

6. KLOTZ.

I. XI.,

.I rch.

Hiochern.

Nioph!/s.

92 (19(i(i). T. 1:. \SI) ~LOTZ, I. 7. tII.GHES, Niochenr. AInaZ. 3, 2ti5 (1956). 8. I
I.

&I.,

IS., J. .lm 9, BESNHOLI).

~~.\LKER,

F.

?\I.,

hr.,

.\NI)

116,

dlelho//s

hV.\S,

I:.

('hem. Sot. 68, 1486 (1940). II.,

.ISU

SC’AUREI~T

J:.,

X.

Gr.5.

Erpil. !Ild. 113, 722 (1943). C., .I r~neit,lit/e/-Po,sch. 4, I, 20 10. WrsI)mtLY, (1950). w., .llnkro~rloz. C’hern. 11, 131 11. s(‘tIoLT.\s. (l!kx3). 12. S;.\ITO. s., liolloitl %. 164, 19 (1957). I. hr., .\ND STKYKER, \‘. It., .r. al,,,. 13. ~LOTZ. C'hcrtr. SK. 82, 5lCi9 (1960). l’., .\sI) Frc.\xs, II. l’., J. .t,,l. 14. RIOLYSEI~S, C'hetn. Sot. 83, 31ti9 (lY(i1). I. hf., .\ND AYERS, J., ~ulpl
l(i. Ii. 18.

19. 20.

I. >I., .\sI) I:L-SELL, J. Vi’., J. J’hys. Chetn. 65, 1274 (19til). I. AZ. ( .1. . I )?1.Cl/et,!. sot. 68, 2P99 (1946). 22. KLOTZ,

21.

I~LOTZ,