150
H. IC. SCHACHMAN AND \\-. J. XACZMAXN
T'ISCOSITI' AKD SEDTI\IE:STATION STUDIES ON TOBACCO MOSAIC S'IRUSl H . IC. S C I I . ~ C I I S I A S.~k N D Doparfvieiil
0.T
w,J. r\AuzRI.ixx
Clicnii.Flry, Priiicctori C'riiucr,si!y, Priricciori. .Vric .JCIXC!/
Rccciccd d u g u s t 19, 1948 INTRODUCTION
It has been found that specimens of purified t,ohacco mosaic virus prepared by different investigators at different times and even by t'lie same investtigat80rat different times show varying physical properties (4, G, 7, 12, 24). The most striking variation is in t)hestate of aggregation of the rod-like particles as revenled by viscosity studies and electron micrographs. Viscosity mcasurements can be used as a criterion of aggregation because the particles aggregate end-to-end t o give higher axial ratios, leading t o an increase in the viscosity of the solution (7, 18). It therefore seems important t o study t,he effect on aggregation of different methods of preparation of the virus and of the physical and chemical environment on the purified virus. This paper presents a stiidy of the aggregation effect, of phosphate buffer during the purification and storage of the virus. Conditims have been found which lead t o the isolation of tobacco mosaic virus in a state of minimum aggregation. Having found these conditions, it seemed pertinent t o use this essentially monomeric virus in order t80study furt>herthe dependence of sedimentation rate on virus concentration and its relationship t o the viscosity of the solution. I n addition, a comparison of the experimental results \vit,h t,he theoretical basis of sedimentation of large particles seemed advisable. EXPERIMEKTAL
Both the common tobacco mosaic virus and the rib grass starahwere used in these studies. I n all cases the virus was concentrated and purified by a series of cycles of alternate high- and low-speed centrifugation (20). Four cycles were generally sufficient t o yield preparations of reasonably pure virus solutions. Preparation I of the tobacco mosaic virus was obtained by one centrifugation in the Sharples supercentrifuge at 50,000 R.P.M., followed by three centrifugat,ions in a vacuum-tj-pc, air-driven, quantit,y centrifuge a t 24,000 R.P.M. Details conc,erning buffer concent'rations are given belo\r. The rib grass virus was isolated in a similar fashion. Sedimentation studies ivere carried out in a Bauer-Pickels air-driven ult'racent8rifuge(1) equipped with the Svensson (22) optical system. Ostwald-type viscometers having a capacit,y of 1 cc. of solution were employed for the viscosity measurements. The viscometers were constructed of a capillary 1 Presented a t the T\rent)--secoiitl Nntionnl Colloid Yyinposiuin, which was I i ~ l t lunder the auspices of the Division of Colloid Chcmistry of t h e ,irncricnn Chemical i3ociet)y ttt Canihridge, Mnssachuset ts, June 23-25, 1948. 2 Junior Research Fellow of the K\'ntional Inst,itute of Health n t the Depnrtnient of Aninial and Plant Pathology, The Rocliefe,llei, Institute fov hIcrlicnl Rcscai~ch,Pi,incctoii, Jersey.
151
VISCOSITY k Y D SEDIMEKTATION O F TOB.4CCO 1IOYAIC VIRL-S
90 cm. long and about 0.06 em. in diameter so as t o yield a relatively low mean Jrelocity gradient of the order of 300 set.-' in one case and about 1GO see.-' in the other. The reservoir h l b of the viwometers possessed a uniform cylindrical ci,osi-iection such that the addition of more than 1 cc. of solution caused a calculnble reduction in velocity gradient. It w:ib possible in this way t o lower the mean velocity gradient in one viscometer t o about GO see.-'
Isolation stiiclics 111 view of the different results obtained by different invest'igators on virus isolated by various methods, it, seemed advisable t o conduct preliminary studies t o determine the correlation het'jreen the soh7ent system used foe the isolation and t'he degree of aggregat,ion of the 1xesult:mt virus. I n accordance with t,he geneid pi.ocedure developed a t the Rockefeller 1nstit)utefor Medical Research in Prinvet'on, Ken. Jersey (20), disodium hydrogen phosphate ]vas added t o .the gi~oaiitl-irpplant mash in an amount sufficient t o bring the expressed juice t o pH T.4BLE 1 T-iscosilics a n d yields f o r diferent victkods of preparation of tobacco mosaic virus PREPARATION
1
C E S T R I F U C \TION PROCEDURE
YIELD O F VIRI,.S P E R C U B I C CENTIUETER O F P L A N T JUICE ?If g
h ., , 13. . C .. D . .. , E, . F, ,
,
.,... , , ., ..... . . , . ., ,
, ,
., . , ,' . . ... ..,. , . . , . . . ... . . , . .'
,I
Three times From water Three times froin 0.001 j11 phosphate Tlirce times froni 0.01 J I phosphate Tliree times froni 0.1 Af pliosphnte Three times f r o m 0.5 Jf phosphate ?'\rice froin 0.1 >IIpliospllate; t ~ i e nonce from \rater
.
1.2
1.3 1.3 1.5 1.7
1.5
7 . A f k t the fiixt high-speed centrifugation u.hich separated the virus in the form of jcll>*-lilcepellets from the I)ulli of the plant juice, the tubes containing the virus pellets ivere tli.l-itletl into sis gixoups u.hich \\'ere then t,reated in different ways. Tnlile 1 s h o w t,hc results ohtnined. Column 2 in the table gives the solvents used t o dissolve the \.irus pellets and the number of high-speed centrifugations f i m i t,hnt, solvent. I n all cases the find virus pellets weye dissollred in the solvent employed in the final centrifugation. The degree of aggregation of the virus can be inferred from the viscosit,ies of the solutions of the purified viruses, In column 3 ni'e slio\vn the values of vS,,/Cfor each virus sample dissolved in the appropriate sol1,ent a t a viius concentration of about 0.2 g./100 cc. The yield of purified virus per cubic cent8imeCerof original plant juice is given in column 4, A4iccoidingt o the Simha (19) equation and the knonm size and shape of tobacco mosaic virus particles (a rod 280 mp long by 15.2 mp in diameter, as shown by electron mici.ographs (10, 18)), [9]should be about 25 cc./g. It is likely that egated virus will not be oriented appreciably at t'he shear gradients ob-
taiiied in the viscometers used here. On the other hand, aggregated viru;; pat,ticles will be oriented ahd t,he meamred viscosities would be lower tJhant'hc t8rue viscosit,y. I n considering t,he xdues of vsl,/C present'ed in table I , it must also be borne in mind that, they \\.ere obt'ained a t concentrations of about 0.2 g. 1100 cc., and thejr are therefore higher than tmhctrue int,rinsic viscosity. I n order t o tleteimine whether the high viscosit,y of prepzwation X \\-as carisrtl by aggregation or t8heelectroT%cous effect, its viscosit,y \vas measured at, different phosphate conrentrat,ions. The results are given in table 2. They clearly shmv t h a t the high viscosity of the virus solutions is not, due t o the elcrtroviscous effect. These solutions exhibited considerable birefringcnce of flo\\., and the relaxation t,imes estimated visually indicated that, tJhe particles \rere much longer than monomers. When the solution in 0.0 1 31 phosphate \\'as rcesrtmined after 120 hr., t'he value of q s , , / C \\asfound t,o h a w decreased from 89 t'o only 81 cc.,/g. It thus appears that the virus isolated by re,pcat'etl centrifugation in water is considerably aggregated. This aggregation is p r o h h l y t80a large cstent irrewrsihle. TABLE 2 Values of qsp/C at d i $ e m n f p h o s l ~ h u f econcetLlralions for ltie i'urL0u.s pwpuvutions pH = 6.9; virus concentratioii = 0.4 g./lOO cc.; qsJC i n rc./g.; phosphate concentration i n inoles/liter I
PREPARATION
PEOSPEATF. CONCENTRATION
1
_______ 0
__--
i
0.01 N
A. ..................... I B .......................
c .......................
11s
,
s9
I
G7 41 32 39
1
F. . . . . . . . . . . . . . . . . . . . . . . . l Aged juice . . . . . . . . . . . . . . .
~
'
70 63
i
0.1 M
0.5 A f
-_. - 8
~~
I
,,
121 109
127 ~
106
98
101
1
115
The results of similar experiments oonducted on preparations R , C,, and F are also shown in table 2. I t is of interest that storage of preparation B in the 0.01 M and 0.1 M solutions produced opposite results. The solution stored in 0.01 iM phosphate decreased in viscosit,y by 5 per cent in 24 h r . , \rhereas that stored in 0.1 M phosphate increased in viscosity by 7 per cent in the w m e period. In neither case did vaJC fall tto 27-28 cc./g., the values found hy 1,auft'er (7) m d Schachman (15) for unaggregat'ed virus. St'orage of prepnrat,ion F in 0.01 M phosphate for 96 hr. produced no significant cliange in viscosity, wherens t,he solution in 0.1 Af phosphate appeared t o decrease slightly in viscosity. I n an effort t o determine the effect of storage of t81ieplant juice, a. sample brought t o pH (3.9 by iiddition of disodium hydrogen phosphu18ewas allowed t o stand at 4°C. for about, 4 \reeks. The virus \\.as then isolated folloiring the procedure used for preparation F. T n the last, row of t,able 2 are presented the viscosities of this virus preparat,ion. That, t81tesevalues are not significantly different from those obtained on preparation F suggests that little aggregation occurs on st,orage of the plant juice before t,he Yirus is isohted.
VISCOSITT AYD SEDIMENTATIOS @F 'YOB.4('('0 hI@h.\I('
T'iscosity
\.111LX
153
StlldiCS
I n vie\\. uf the r ~ s i i l t ~ol)t:tined s in different, sol\.ents ant1 Lerarise of the sjlisceptibility of t,he viins t o aggregation, it seemed woith\\.liile t o in\.estigate in greater detail the \.iscositieli of soliit ions of purified \.iriis. The common strain of tJobacco mosaic: J+iis (preparation I ) :i,ncl t8heril) g i ~ sts rain \\.ere isola'tetl according t80the procedni-c used for prepa,ixtion F. This inetliotl \\.as used be(lause t,he \virus appeai*etlt o consist, principally of niononier \\Then tmhesoliit'ions \\westmudiedin 0.01 111 phosphate, :und nlso 1)ec:iuse t h e final solution cont8ainec1 \.ei*>* little salt, Thew soliit ions \\.ere c~onsitlerecltmohe \\.:ttw solutjions oC iJhe virus, alt,liuiigli il t i w e of salt is nc.ti~allypiwrnt. Figure 1 sho\ys n plot of qSt,/cI21s. C for piqinixtioii I in 0.01 A1 phospliate and 0 . 1 nf phospha,tc%. .As, in the rase of thc p i q ~ a r ~ ~ t ~Iistcd i o n s in t:tl)lc 2 , n high
-
50;_.-----illl_p:
40
' : . :30I :
Y 3 2010-
o
u
A
0.1 M phosphate.
0
Freshly isolated virus in 0.01 M phosphate.
0
5 months old virus i n 0 01 M phosphate.
i3
viscosit>' is oht,ainetl in t'he more (~oiic*ent ixt'etl ,buffer. It is of iiit,ei*estthat, storage of pui3ied virus in \vatel. for fire months did not' in itself m u s e appreciable egat8ionof the iviiws, for the i.isco;;il ies of t81ica,ged i-iiaiis in 0.01 111 phosphate are almost t h e same tis those of t,lie freshly isolated virus. \\'hen aged virus was dissolved in 0.01 31 pliosplia,tethere was il barely detect8ableinciwse in iiscosity \vith time. On t,he other ha,nd, \rlieii t h e iiged virus \\.as dissolyetl in 0.1 A 1 phosphate tl1ei.c \\.:is n, \-ei-yrapid, i f not' jniiiiedia,te,iiiciwse in viscosity, giving values of ?,,,jC grcnter than 110 cc./g. This difference in l)eha\-ioi*;It, 1 he t,\vo buffer (wncentrniions is also found for fresh \ T i i w , hiit here t'he yisrosities in 0.1 -31 phosphrtte nine m u c h lo\vei- uncl c h a n g ~only slo\\.ly \\-it11t'inie. One might snspect f i ~ i n iprevious \\.or*li( 1 3 ) that tlic iippei' ( * i i i . \i~n figui,e I c of 27 ('c..,'g. as c' ~ l p p l ~ l . ) : w !zero, le~ h i t n u cle\\~onlcla p p l ~ o n ~;I~ \~allic h of tailed stud!. in this coiicenti'ation range \Y;LS macle in the present inr.estigat ion. An attempt \\.as made, Inon-el-er,t o eoiinter t h e aggreg:it ion effect o f the more concvmtixtcd huffei. 1)). i,nising the pH of the sohition. 111 :I 0.1 JT phospha~tr
154
H . K . SCHACHMAS L Y D \ I . J. IiAVZRIANS
nolutjion of the aged virus at p H 8.2 the initial value of q8,]/C \\.as 72 CY.,g., instead of about 110 cc./g. observed at p H 7 . Moreover, there was a detectable decrease in viscosity with time, resulting in il. value of 51 cc./g. at the end of 40 hr. Dialysis of the stock viyus solution against 18 1. of distilled water brought to p H 7 by the addition of sodium hydroxide raised the viscosity by about 30per cent. This was probably due t o the increased electroviscous effect caused by removal of the small amount of electrolyte still present in the original stock virus
0 I 0 6 rng/cc 0 2 13 mg/cc
A
90
4 92 mg/cc
A 9 8 4 mg/cc
80 70 -
=u ? .-Z 60-
< 6 50-
40 30 -
FIG.2. Dependence of the viscosity of virus solutions on salt concetltration at pH 7
solution. Upon addition of phosphate t o make the dialyzed solution 0.01 31, the viscosity dropped t o 28 cc./g., thus showing that the dialysis a t p H 7 did not cause aggregation. When the dialyzed solution was made 0.1 ill in phosphate ~ s , , / Cincreased t o 113 cc./g. Detailed experiments on the rib grass strain are presented in figure 2, Ivhere values of vLp/Cfor four different virus concentrations are plotted against the buffer concentration. As with the other virus samples a pronounced minimum is obtained in about 0.1 ill phosphate. In all measurements the viscosity values are the averages of five successive readings obtained shortly after the dilution of
VISCOSITY AND SEDIMENTATIOK O F TOBACCO MOSAIC VIRUS
153
the virus st'ocli. On the right-hand side of the curve the readings increased slightly during t'he measurements. Extrapolation to ze.ro time is difficult , hecause all the solutions were centrifuged at low speed immediately after mixing t o remove dust pnrticles. The time i n t e n d T\-as short enough, however, t o indicate thnt on mixing n-ith 0.1 AI snlt there must have been a very rnpid, if not immediat'e, increase in Tkcosity ivhicli can be attributed t o end-to-end axgregation of t'he virus part icle.;. Experiments in \\-hidl the electrolyte \vas potassium chloikle inst,eacl of phosphate yielded similar results. A compai-ison of t,lie effect of chloiide ions i3.s. phosphate ions slio\ved the interesting result t'liat at a virus concent1,ntioii of 0.21 g.,'100 cc. qsl,,iC in 0.1 Ji potassium chloride i\w 110 cc./g., whereas in 0.1 :lI p1iosphaRe it \\'as i 1 cc.,'lg. Tlie potassium cliloi,ide solut'ions were unl)uflei*etl 2nd their pH \\.as about 6.1. Therefore this liighr~r viscosity cannot lie attiibuted solely t o the chloride ion, hecause aggreg,ziioii occurs more estensivelj* at. low pH. In nn effort t,o provide atltlit~ionwlei.idence that the increase in viscosit,!. a t high salt concent rations is due to end-tjo-entl nggregation some experinienl s irere done at i-ai.j'ing shear graclient,s. There \\-as no detectable increase in viscosit,y of a dilute rvirus solut,ion (0.11 g./100 cc.) in 0.01 171 phosphate \\-hen the avemge shear gradient \vas rediiced from 300 sec.-' t o 150 sec.-l On t,he other hand, the virus solution in 0.1 -11 phosp!int'e showed an increase of ahout 20 per cent in vsp/C in going from the high t o the low shear gradient. This is proliably because the monomers are almost, randomly orientled a t even 300 set.-', whereas the aggregated virus part8iclesare partially oriented at this shear gi-adient. When the shear gradient is ieduced, the orientation of the longer part'icles becomes more random \ritJh a, consequent increa.se in viscosity. An even more dilute solution cont.aining 0.053 g. \+.us/ 100 cc. in 0.01 111 phosphate \\'as studied at shear gradients from 300 sec.-I t o about GO set.-', and again no detectahlc change in v S J C was observed. Tlie average value obtained was 29 cc./'g. I t is, of course, necessai-yt o measure Jiscosities in a Couette-type viscometer before it can lie finally concluded tJliat values of aliout 27-28 cc./g. represent viscosit,?r values undcr conditions in which the part'icles amresubject t o completJeBra\\-ninn m oT-ement. SccEi,nct~iatioristudies Figure 3 shows t,he dependence of sedimentntion constant, on virw concentration for preparat'ion I. The secliment,at,ion clats Irere reduced, according to S\,edl)eig and Pedersen (21), t o a stnntlai'd state coiwspontling t o n,nter at 20"c'., where the i-iscosity correction is for tjhe viscoait'y of the solvent. There does not, seem t o be any significant)differencein the sedirnentnt'ion rates of tobacco inosnic virus in 0.01 111 and 0.1 11 liuffeix. This indicates thnt 0.01 11 phosphnte is sufficient t o eliminate the priniai,jr charge effect v.hieli, it' present, would lend t o loirer sedimentation i,ates in t11c solutions of lo\\. ionic strength. The data for the espei'iinents in 0.01 d l pliosphate are i*eplott,ed in figlire -c and, in acldit'ion, t h e sedimentat ion constant corrected fol, t,he viscosit,y of t,lic: soliition through miilt,iplication by 7 , '70 is plott,ed against concentrnt,ion in t,Iic;
156
H. I(. SCHACHMA?? AND U'. J. K A U Z M A "
same figure. It is readily seen that the product Xv/qo is constant over the concentration range studied.
and
loob
'
d.2
'
64 ' 0!6 ' 0!8 ' 110 ' b!2 Concentration in g./ 100 cc.
'
114
'
l!6
'
1.L
Fro. 4. Effect of solutio11 viscosity on the sedimentation constant oE tobucco mosaic virus
Though the corresponding data for preparation I in 0.1 ill phosphate are not plotted in the figure it is easily seen t h a t they lead t o overcorrection of X. The S vs. C data for the virus in the two buffers shorn no significant differences
\\-bile, t ~ bslio\\.n in tiguw 1, the \*iswsity is Iiigher in 0.1 J I plivspliat,c. Theref'oite, the pi.oc1iic.t of S : ~ i i t l7/70 in 0.1 J I pliospliatc \\.odd give values \\.hicli are la,rger than the (~orrespoiicliiigprodiict in 0.01 .I1phosphate antl \vhic*liincimease \vith inci*ensing coiicent'ixtion. Further setliment:ttion st8udieson solutions of tobacco mosaic*virus cont8:iining varj'ing amoiints of sucrose \\-ereperformed in an efioit t o pi*nvitle:itlrlitional informat ion on the clependence of sediment:ttiori i~at'e011 virus concent8rnt8ion. I n t,hese experiments both the J-irus antl the sucrose c:oncent,i.nt,ionsin 0.01 dd phosphate :it pH 7 \\-ei'e1-arieclso as t o produce diit8ionsof constnnt, oi.ei--nllviscosity. The ixesults are presented in table 3. C'oliimris 1 and 2 give the T-irus and sucrose concenirat,ion, respectively. In column 3 a r e @-en the ohser.i-ed sedimentairion const8ant8s, and in coliimn 4 :ire giT-en t h e sedimentation const:int's calculated according t'o Svedherg and Pedeixen (211, where t,he solvent 1-iscosity is t8hat of watJer plus t8he dissolved salts and sucrose. The densitmycorrection is made using 0.788 cc.,ig. for the p;ii,tial specific \ d i m e of t h e v i i w . This \\:as used TABLE 3 \'IRUb COhCEUTRATlOK
g
SUCROSE
1
_-p--l
/roo cc 0.11 0.30 0.63 1.04
1.58
>
CONCFNTRATION
-
~~
I
6 /IO0 (C I
14.3 12.4 9.i
5.6 0
116 118 122 121 115
vim
s>0
__
171
1
169 160 143 117
-
-
57/70
-
~
___
I
1
1
1
1.539 1.537 1.56 1.57
178 182 191 191
1.547
181
because the hytirrttion atudies of Schavhnian and Laufl'er (1ti) indicate that the density of tobacco mosaic \Tirus in sucrose solutions is about 1.27 g./cc. Columns 5 and G give 7/70 and S T / ~ respectively, O. \\-liere 70 is the viscosity of water and 7 is t h a t of the virus-sucrose solution. I t can be seen that approximate constancy of S7/70 results. DISCUSSION
In considering the results of the isolation experiments given in table 1, it is important t o realize t'liat,the yields of purified virus per cubic centimeter of p h i , juice vary appreciably. This could nieiln that, in those solutions giving a high yield, such as 0.5 ill phospliat'e, some protein ot'lier than virus protein is included in the final preparation. If this is true, then of course the viscosity measure-. nients do not give a true picture of the state of aggregalion of the final prepara-, tion. On the other hand, t,he yield in water could be lo\v because of incomplete sedimentation. It is well kno\vii that) sedimentation of concent,rated virus in water solutions is more difficult, presumably ou,ing t'o the electrostatic repulsion of the virus part'icles. The pellets obt>ainedin water solution are much larger and less firm than those obtained in salt solutions. A coniplete ansn-er t o this quest,ion would require measurenients of biological activit
