5G8
,JACKH. TREMAINE AND MAXA. LAUFFER
TIIlS C‘KiIiGE EFFECT I N SSEDII\lF,KTSTIOS1 B Y JACK H.
T R E M A I N E 2 f 3A S D AXAX
A. LAUFFER
Department of Biophysics,’ University of Pittsburgh, Pittsburgh
is,Pennsylvania
Rerezved August 28, 1969
A detailed experimental study was carried out on Southern bean mosaic virus (SBAIV) for the purpose of investigating the charge effect in sedimentation. This material was chosen because its particles are essentially spherical: i t is moderately cltahle; it has a relatively large sedimentation rate; and it can be obtained without extreme difficulty. The effects of p H , ionic strength and concentration on the sedimentation rate were studied. Auxiliary measurements were made on electrophoretic mobility and valence. All of these data were analyzed in terms of existing theories of the charge effect. The most widely known equation, that of Tiselius, seriously underestimated the effect otlserved in the present study. The increase of the charge effect with virus protein concentration was linear as predicted. The theory of Booth predicted an unrealistically small charge effect. Furthermore, the prediction of variation of the charge effect with concentration of colloidal materid did not agree with the experimental results.
Introduction effect on the sedinientation velocity of a single The generation of nil electric field when a suspen- particle. Solution of this eqiiation for a qi~~pension sion of glass heads is allowed to settle was observed of many particles is possihle when the sedimenting by D01-11~in 1880. Presumably the negatively boundary is nil infinitely thin sheet. An approuicharged glass beads sediment faster than the mation of this condition might he obtained in the partner cations, estahlishing an electric field in the cell of the analytical ultracentrifuge. The purpose of the present iiivestigatioii was solution. Snioluchowski6 derived an equation predicting to study the magnitude of the charge effect, to the magnitude of the electric field generated by measure the parameters involved in the Tiqelius sedimentation of charged particles. He also noted and Booth equations and in this manner to test that this field would reduce the sedimentation ve- the validity of these equations. The colloidal locity of the charged particles, and derived an particle employed was chosen for several reasons. The secondary charge effect, which i.: independent equation for this charge effect. Tiselius7 derived an equation for the primary of the sedimentation rate of the colloidal ion,* was considered to he small in comparison with charge effect in sedimentation the large primary effect attrihutahle to the high sedimentation rate of SBMT‘, the particleq of which are known to he essentially qpherical. Informawhere s’ is the sedimentation rate of the charged tion concerning the sedimentation rate, partial particle, and s is the sedimentation rate of the un- specific voliime and molecular weight is availcharged particle, u and 1, are the valence and elec- able. l o n l l trophoretic mobility of the particle, n is the conMaterials and Methods centration of the particle in molea/ml., F is the Preparation of Concentrated Southern Bean Mosaic Virus faraday constant, and ks is t.he specific conductivity (SBMV).-The virus nucleoprotein was prepared from inof the solution. The equation was found to agree fected Phaseolus vulgaris 1, variety Bountiful bush bean with experimental data obtained with phyko- plants which had been ground and frozen a t the time of harvesting. erythrin. The purification procedure was adapted from the centrifPedemens studied the charge effect on the sedi- ugation procedure of Price12 and the chromatographic promentation rate of egg albumin and of bovine serum redure of Shainoff and Lauffer.13 Plant juice expressed albumin. He found that the primary charge from thawed material was clarified in a Serval1 angle centrieffect was inversely related to the conductivity fuge (15 minutes at 5,000 r.p.m). and then concentrated in a model L centrifuge (three hours) a t 20,000 r.p.m. in a of this solution and directly related to the concen- Spinco KO.20 rotor. The pellets were resuspended in 0.1 ionic tration of protein, as predicted by the Tiselius strength phosphate buffer a t pH 6.7 and then subjected to equation. two cycles of alternate clarification and sedimentation. A By a consideration of the dipole field which dark brown pigment was observed in the virus preparation after this centrifugation procedure. The pigment was rearises from a distortion of the ionic atmosphere moved from the virus preparation by column chromatogof a charged particle when the particle is sedimented raphy. A column of Amberlite CG 400 ion-exchange resin away from its ionic atmosphere, Boothg derived was adjusted to a pH of 6.70 with a buffer composed of 0.008 an eqiiation predict>ingthe magnitude of the charge iM phosphate and 0.064 M sodium chloride. The pigmented (1) Presented a t t h e 131st meeting of t h e American Chemical
Society, Miami, Florida. April 7-14, 1957. (2) Canada Department of Agriculture, Science Service, on eduostional leave-of-absence during t h e rourse of this work. (3) Abstracted from a thesis submitted b y J. H. T. t o t h e Gradua t e School of the University of Pittsburgh in partial fulfillment of t h e requirements for t h e degree, Doctor of Philosophy. ( 4 ) Publication KO.73 of t h e Department of Biopnysics. (5) E. Dorn, Ann. Physik. 10, 46 (1880). (6) RI. V. Smoluchowski, Uraetz Handbuch der Elektrizitaf und des Magnelismus Leiprig, 2, 385 (1921). (7) A. Tiselius, Kolloid Z., 69, 306 (1932). (8) IC. 0. Pedersen, referred t o in “ T h e Ultracentrifuge,” Oxford University Press, 1940, p. 26; THIBJOURNAL. 62, 1282 (1958). (9) F. Bootli, J . Cliem. Phys., 22, 1956 (1954).
virus preparation was adsorbed onto the column, and a white virus solution was eluted with the buffer, leaving the pigment adsorbed to the column. Before each sedimentation coefficient determination, the virus solution was dialyzed against the buffer used in the experiment. After the dialysis, the preparation clarified by low speed centrifugation, filtered, and the preparation was divided into three samples. One sample vas centrifuged to determine the sedimentation rate of the virus; one uas
(10) G. A . Miller and W. C. Price, Arch. Btochern., 10, 467 (1916). (11) M.A. Lauffer, N. W. Taylor a n d C. C. Wiindc-r, Arch. B~ociiem. Baophys.. 40,453 (1952). (12) W. C. Price, A m . J . Botany, 3 3 , 45 (1940). (13) J. R. Shainoff and A t . A . Laufler, Arch. Diorhem. Biophya.. 64,
315 (1956).
itsed to detcrmine the specific conductivity of the solution, and thr rcm:tining sample was nsed for pII d&rmination tit, 15 minute intervals during the centrifugation of the first sample. T ~ Icomplete the experiment, density and concentration measurements were made on the pooled samples. Ultracentrifugation Studies.-Sedimentation studies on P R l I V nucleoprotcin were carried out in a Spinco Model E ultracentrifiige at room tempcrature. The tempcrature of each run %as taken as the mean of the initial and final temperatures of the rotor as determined with a thermocouple. The difference was usually less than 0.5'. Sedimentation rates mere vorrwtrd to a standard state, s2%u,by the usual method employing an apparent partial specific volume of O . i O O reported by Lauffer, et al." Density and Viscosity.-The densities of all the solutions studied in the ultracentrifuge were determined with a 2-ml. pycnometer a t two temperatures above and below the average temperature of the centrifuge run. Viscoaitits of the solvents were determined in an OstwaldC:mnon vihconieter. The viscometer was calibrated with water at 20.25 and 30' and the relationship determined that 9 = 1. T i 3 Y 10-2 dt, where d is the solution density and t is t,he outflow time. The viscosity of the solvent and densitv of the solution at the tempt.r:i,tu"reof the centrifuge run were determined by interpolation from measurements above and below that temperature. pH and Conductivity.-The pH measurements were made using a Reckman glass electrode model G pH meter. These measurements were made at room temperature on one sitmpie of virus solut,ionevery 15 minutes during the centrifiigat,ion of anot.hcr sample. After the centrifugation of the first sample, the pII of the virus solution in the centrifiige cell WAS mcasiired with a one-drop electrode assembly. The pII vdue used in thr: cslculations was the arithmetic mean of the pH values fount1 for all the measurements of both samples. The pH measurements made on the virus solution in a single run never varied more than 0.07 of a pH unit. The fluctuation in the pH measurements produces rather large differences in the product of valence and electrophoret,ic mobility, hut greater pH control cannot be obtained easily with low ionic strength buflfers because of the low buffering capacity. Conductivities were measured on other samples with a Shedlovsky cell and a model RCIB conductivity bridge, ninnufactured by Industrial InstrumentB, Incorporated. This appar,ttus was calibrated using standard KC1 solutions and has a cell constant of 12.20 cm.-'. The conductivity measurement,s have an crror of less than 3%. The conductivity of the solution a t the average tempernture of the centrifuge run was interpolated from the values at temperatures above and below. Electrophoretic Mobility and Valence.-The variation of electrophoietic mobility with pH of SBMV solution a t 0.002 ionic strength was determined in a Spinco electrophoresis apparatus. Samples having a nucleoprotein concentration of approximately 0.7 % were dialyzed for a t least 24 hours with two changes of buffer. Subsequent conductivity measurements sliowed no significant differences between snmples and dialysate for any of the samples. Conductivity measurements were made at 25' although the electrophoresis water-bath was maintained a t 0.9'. The mobility is reported at 215' since sedimentation rate determinations were nude at room temperature. Watanabe, et al.,I4 found an error of spl~roximately10% by the use of this procedure with horse serui:i albumin a t p H 7.7. The pH was determined :It, rooni tc:nil)eratnre (iipproxim:Ltely 25') with a Beckmail Alodel G p1-I mrti:r. The virtis \vas titrated t o determine the valence of the riucleoprot,8inin the p H range employed in sedimentation studirs. The v i x s solution was dialyzed five days against distilled water with three changes of dialysate. The virus solution w a ~rumov-d from tlie dialysis bag and split into three 5-ml. aliquots. The p H of one of the 5-ml. aliquots was measured after the addition of 0.1-ml. portions of 0.01 iV NsOH. This procedure .was repeated with another 5-ml. aliquot employing 0.01 LV HC1. The remaining aliquot was employed to determine the concentrat)ion of the virus (grams/unit vollime) by rr,easuring the refractive index differences between (14) I. W i t m a b r , 1306 il950).
iu'. Ui and >I. Nakzniura, Tars JOVRNAL,
64,
the virus solution and the dialysate. An additional titration i n 0.003 ionic strength NaCI W~LScarried out in the same manner. The number of OH- or H + ions bound per virus particle was calculated by the following method. The concentration of SBIlIV in moles/liter [VI was determined by employing a molrcn1:tr wc,ight1lof 6.1 X IO6. The eoncentration of free OIf- or [OHIF (moles/liter) was calculated from the ohserved pH. The concentration of free and botind OH- or p(OH) = 14.00 - pH = -log [OHIF" [OHIF + B (moles/liter) was calculated from the volume ( V,) and titer, 0.01 N , of added NaOH and the total volume of virus solution after addition of NaOH ( Vt). O.OlV,
[OHIF+B
=
vt
The base bound [OH]= moles/liter was determined by siibtrac*tirig [OII]F from [OH]F B. The number of OH- ions bound per particle was obtained from the base bound and the molar concentration of virus present A similar method of calculation was employed in the acid titration. Determination of Virus Nucleoprotein Concentration.Virus concentrations were determined by differential refractometry. The refractive index difference between the virus solution and the dialysate was determined for each experiment. The concentration of the virus in the solution in g./100 ml. was obtained by dividing this refractive index diffrrence by the specific refractive increment, i.e., the refrartive index difference between the solvent and a solution rontaining 1 g. of virus per 100 ml. of solution. Two determinations of the specific refractive incremrnt were made on the virus preparation. Values of 0.00176 and 0.00173 (100 ml./g.) were found. A value of 0.00175 was used during the present study. The results are in good agreement with the specific refractive increments of nearly all proteins and of nucleic acids, which were found'? to lie within 10% of a mean value of 0.0018.
Results Variation of Electrophoretic Mobility with pH.A study of the variation of electrophoretic mobility with pH was made for SBNV using phosphate bufTABLE I THEMOBILITY OF SBMV IN 0.002 TONIC STRENGTH PHOSPHATE BUFFER^
P€1
5.60 6.00 G.28 6.45 6.50 6.75 7.20 7 .%50
Electrophoretic velocity, cm./sec. x 10-4
S1.36 0 -3.07 - 4 . $19 -6.18 -8.80 -11.65 - 13. I S
Field strength, v./cm.
Mobility, 2.rl c, 22 . 6 2(i,0
15 17 18 20 21 7 8
TABLE I11 EXPERINENTAL AXD PREDICTED
Tnitial virus nucleoprotein concn.
I"
14
OF THE
d.
,
2-- , 0
2:;. 0 2 i ,7 2 ; .1 2 ; .:3
PH
7.00 6.70 7.46 7.20 7.10 7.00
6.90
2-1.7 24.0 20.5 26.8 2,;. 9 2ti. I 21;. 5 2.5.3 23.5 21.0
6.63 6.25
g./lOO
ml.
1.81 4.90 3.97 3.79 1. G S 3.91 3,38 2 ,8!) 3.70 '4.0'4 3.10 2.65 2.07 1.57 0.96 2.90 2.77 3.69 3.75
Specific conductivity mho/cm. x 10-4
50.0 50.0 1 .9:3 2.14 1.47 1.64
I .74 3.5'' 1.87 1.50 I .64 1.95 1.94 I .78 1 .6(; 3.74 2.62 1.67 1.66
CHARGE
ate cm.3
103.8 95,o 70.8 74.4 91 .I) 67.2 74,2 85.7 74.7 69.9 79.6 86.1 90.9 93.2 102.8 88.2 8G. 0 80.3 95.2
a
om.* v.-see. x 104
104
524
893
4:
