R.%BBIT L I V E R SUCLEOPROTEIS
207
ELECTROPHORETIC -1SD ULTRACESTRIFUGAL I S V E S T I G A T I O S OF R-iBBIT LII-ER SI-C'LEOPROTEIY Q. \-.ISX I S K L E Depcrrtment
OJ
ASU
WESLEY G . FRASCE
C ' h e m z s t r y . The O h i o S t a t e l T n i r e r s i t y ,ColumFus, O h i o
Receired d i i g u s t 25, 1947 ISTRODUCTIOS
Sucleoproteins are known to be intimately associated with such biologically important entities as the chromosomes, genes, and viruses and have received a considerable amount of study in connection with virus diseases and cancer. The naturally occurring tissue nucleoproteins which are found in the nuclei (chromosomes and genes) and cytoplasm of normal plant and animal cells have not been as intensively studied, however. Only calf thymus nucleohistone has been characterized n-ith regard to its molecular weight, particle shape, and electrophoretic behavior (2, 3, 5 , 6, 7). This leaves a large and important field open for further investigation. For this investigation, it n-as decided to prepare rabbit liver nucleoprotein by a mild method, determine the homogeneity of the preparation by means of electrophoresis, sedimentation velocity, and diffusion studies, and obtain information concerning the molecular weight, particle shape, and electrophoretic mobility of the liver nucleoprotein. EXPERIJICST.IL
PROCEDCRES
Electrophomis m easzircinen ts Electrophoretic studies n-ere made bi- the moving-boundary method, using a Tiseliuq electrophoresib apparatus and standardized techniques Ivhich have been described by Longsn-orth and JlacInnes (11, 13). Sedi7nenfaiion-vclocity measrtrements -1n air-driven, vacuum ultracentrifuge of the Beams type was used for sedimentation-velocity measurements on rabbit liver nucleoprotein. This ultracentrifuge has been described elsen-here (10). The sedimenting boundaries were follon-ed by means of the schlieren bands lvhich were formed in conjunction with a long-focal-length schlieren lens and a knife edge. For a fen- of the ultracentrifuge runs, the Lamm scale method (9) was used to record the concentration gradients in the sedimenting boundaries. Scale photographs were taken through G mm. of solution with a scale distance of approximately G em. -111 measurements were made at a speed of 30,000 R.P.JI., corresponding to a centrifugal force in the center of the cell of approximately 60,000 times gravity. Sedimentation constants ivere calculated from the equation,
1 Presented a t the Twenty-first Satiorial Colloid Symposium, which was held under the tuspices of the Division of Colloid Chemistry of the American Chemical Society at Palo ilto, California, J u n e 15-20, 1047.
and were reduced t o sedimentation in water at 20°C. iJy meuns of the folloiring relationsliip :
The symbols used in equations 1 :tnd 2 h a w been atantIarclized by S v c 4 i m g :Ind l'edersen (21). -1partid ipecihc x oliimc of O.658 TI a< awimed for i;iMit liver nucleoprotein. It is the w l u e n-hich ha': hcrn icpoi t c d for c d f tllnnu. n11clcohistone by Carter and Hall ( 3 ) . 1)iLTusiore meuswcnieizts Measurements of tliffii':ion coefficients were matlc in the Tivliu\ r.lectrophoresis apparatui, iizing n standard I 1-ml. cell in ~rhichthe limbs werr diT itlcd into two sections by a central parting plane. Thii: cell v-as mounted ip. :iCpPcial rack which could IF moved ont of the optical path irithout disturbing the tliff'usiiig boundarieh. duch an arrangement made it poshible to perform clectropliorezis euperiments during the intervals between diffusion photograph*. T l i ~ use of the electrophoresis app:u.ntus for diffusion nieahurcment* ha- hrcn described b y Longs\\-orth (12). The diffusion r\perinicnt\ \\ere perfornicd :it 1.0"C'. ,wtl the diffu-ion cwi-t an1 \\-ere calculsterl hy ti\ o tiifferent method\: ( 1 ) the maximum ordinate-area method, and ( 2 the mrthotl nt momentb. The follon ing ielation*hip n:i- used in calculating tlifl'u*ion cun.tantz by the maiimiiin ordinate-urra met hod :
-
ivliere D, is the mcu*ureci tlift'u&n constant, 1' i- tho absolute temper:ttiirc~of The experiment, T~ is the .iiscoaity of the wlrent at temperature T , iind v?ois the viscosity of water at ZO"('. l'RBl'.\l1ITIOS
.\XD CHAIRI C T I ~ I I I Zk T I O S O F S U C L E O P R O T J ~ I S
.Icthod l of prepuTat ion
Three preparation:, of rabbit liver nucleoprotein T \ T ~ ( ' made, using t hc' procedurr given by l\irsli>- and Pollister (16). The ralhity \yere killed by liecapitation and the livers w r r removed immediately and homogenized in :I Waring blendor in the presence of four times their weight of ice-cold saline wlution (0.14 N sodium chloride) buffered t o p H 6.8 t o remove albumin and globulins.
R.\BHIT
L I V E R hX-CI,EOPROTEIS
209
The nucleoprotein, which i3 insoluble in 0.14 J/ sodium chloride solution, remained in the residue. It v :15 subsequently dissolved bj- treating the r e d l i e n-ith 1 JI -odium chloride solution. Purification of the tli.3olved nucleoprotein was ol)tniiird by precipitating it in 0.14 JI sodium chloride rolution and re&.solying the precipitate in tlic 1 A I solution. A 1 solution.; w r e buflerecl to p1-I 6.8. ‘ h e nucleoprotein \]-a\ precipitated :mi diQsolr-edthree times, with all operation3 being carried out in a cold room a t 0°C. The dry weight of nucleoprotein obtained amounted to 0.1 per cent of the wiglit of the o-et 1iVers taken.
Analyses S i t r o g m and phobphorus analyses were made on dried preparations of the livt’i nucleoprotein, using n standard micro-Kjeldahl method for nitrogen and a 0 48t
0.181
c
\
0.121
I
01 220
I
\L
’ ’ 240 260 280 300 W A V E L E N GTH - M ’
‘
?
I
’
32ci
FIG.1. I~ltravioletabsorption curve for rabbit lircr nurlcoprvteirl dissolved in water coloiinx,ti ic. method for phosphorus (1). Tlie average3 of three determinations err: pt=r (cent S = 16.83; per cent 1’ = 4.9i. A-sumiiip that the nucleoprotein derives its phosphorus content solely from iTs iiuclcie :icicl component aiid using 9.91 per cent as the phosphorus content oi the nucleic arid component. The percentage of nucleic acid in the nucleoprotein is eqtiniaTct1 t o lie appro\iiiiritely 50 per cent. The I )i-clic diphenylamine reaction described Ly Schneider (18) and Seibert !19) K I used ~ in a qunlitxti7 e nianner t o identify desoq-ribose nucleic acid in the niicleoprotein. -1 strong positive test was obtained. -Also, an ultraxiolet absorption curve mts o1,tainecl on a sample of liver nucleoprotein which liati been dialyzed against diqrillctl water. The curve, s h o ~ in n figure 1, exhibits the Vharactcristic nucleic acid ahorption masinium a t 2600 A. (8). 11
210
Q. VAX W1XKI.E . I S D K E S L E P G. FRASCE EXPERI3IEST.IL RESULTS
Elcclrophorctic measurcmetits Electrophoretic experiment- 11 it11 rabbit liver nucleoprotein \\-ere performed a t 1 arious pH's in bufker holiitionb of t n o different ionic strength olitwinetl ~ O I , iiiiclr,oprotc.iii \\-llicli rwci\-c(I t\\m prccipitntions, : m l ~ \ ~ l i i c ~TI-as ll dissolveti in 1 -1L d i i i n i c-hloritlc. 0.02 ionic s t ~ ~ > n gphosphutc, th pH T.2. 'I'lic. Ieatling (~ompotic~uts in thc awcntlinp :ml tlescentling pattern!: :trr> iiuclcopi,otein. I - l o \ v c i ~ ~011 r , thv asccnding sitk the niicleoprotein fraction is split into t\vo separate peiikh. .it pH (j.4 no ,splitting of the nucleoprotein peak on the ascending sitlcl \iw o1)s;cri.etI. IIo~\-ei-oi~. at p H 5.4 lmth the ascending :inti the, tlcwentling peaks ,split into t \\-o components. 7'he patterns o1,t:iinetl for niicleoprot cin ~vliichrcceiiwl t ivo precipitations shon-rtl an appreciable amount of hctcrogeneous, slon--moi.ing material t o bc present at :ill pH's studied. Xt pH 3.9, in 1 -11 sodium chloride, 0.02 ionic strcngth acetate bufft~r,the niivleoprotein peak split into a numljer of components, and, in this instance, the fastest component mol-etl with a molility of thymus nucleic acid (see Stenhagen and ,
Teorell (20)). 7 h i s SuggSTS the possibility that nuleic acid has been split away from protein and is migrating as a separate component. In viex of x h a t is already known concerning the salt-like character of the bond hetween nucleic acid and protein in nucleoproteins (4,6, 15), the tendency of nucleic acid t o split away from protein in I 7I sodium chloride solutions is to be expected. The mobilities obtained for nucleoprotein in 1 sodium 'I chloride solutions are listed in table 1 and pIotted as a function of pH, in figure 6. I n table 1, the column An X to4 represents the refractive-index difference between nucleoprotein solution and buffer solution as obtained from the area under the uucleo-
-
7 ASCENDING
FIG.4. Crude solution: 1 \.I sodiunTchioride, 0.02 time, 610 inin. Field strength, 1.31 volts'cm.
DES GEN DI N G
i.1
piiosphate, pH i . 1 . Electrolysis
- ASCENDING
DESCENDING FIG.5 . Rabbit liyer nucleoprotein prccipitatecl t\vo ?imes. Dissolved in 1 If sodium chloride, 0.02 U , phosphate, p l l 7 , 2 . Elecrrolysis t i m c s , L'O9 min. Field s t r e n g t h , 1.61 \TO1 t gicn1.
protein peak of the descending p:itterii. In ail cnse;. the mobilities listed in table I represent anionic migration. For some of the runs in I X sodium chloride solutions a nnmber of nlobilitiey hn\ e hceii listed. correqponding t o the number of relatively fast moving peaks which Twre o b m i ed in the electrophoretic patterns, and, in adtlitioiz, the components u-hicli w r e nccompanied 11:- tlirbiditv hni e been st:Irretl. SedzinoLtation-uelocifU m a Gedinientntion-\-elocitymeasurements were made on rahiit liver n u c i ~ijpi uteiii nhich had bpen prccipitoterl thrre times and nhicli ~ ; i +~li-olvrd in i TI wdiiirn
213
R I B B I T L I V E R NUCLEOPROTEIS
chloride, 0.02 ionic strength phosphate, p H 6.4. Three different nucleoprotein concentrations were studied in order t o determine the effect of concentr at'ion on the sedimentation constunt. R y means of the scsle method, the sedimenting houndary v a s found to contain possildy three closely spaced peaks or components. The sedimentation data recorded in table 2 are averagr values for
s
5 0
> 0 W
x
>-
t d
m 0
H
-2oi
1
4.0
I
5.0
I
6.0 PH
7.0
I
8.0
F I ~ I;.. >~lectrophorcticinobilit?. w m s p l f . C'urvv -1,rabbit liver nucleoprotein i n 1
.TI sutliurn cliloride buffer solutions; c u r v c 13, rabbit liver riucleoproteiii i n 0.02 p phosphatr I,uffer solutions; rurvc C'> t hyinns nucleic acid in 0.1 p 1)uffcr sulutioris (froin Stt.nIiagPn anti Trori~ll I ' . 1 , w
--
J\CE\TR*TlrO\
-
-
DE P K O T T I \
0
Ill1
0 22 0 11
__
-~
--
__
I
0 00
0 00
-
__ ____
510
Sledbercs
IO 4 15 4 16 5 28 3*
________.
* l.\tr:+pulared \ d u e . the group of peaks. The extrapolation to zero concentration was made by plotting 1 versus concentration.
Diflusaon measurements Diffusion nieasurements were made in dilute phosphate buffers and in 1 JI sodium chloride solutions, using rabbit liver nucleoprotein TThich had been precipitatetl three times. These measurement- also showed the existence of
a molecular. inhomogeneity and the inhomogeneity \\ab greater in 1 ;li bodiuni chloride solutions than in the dilute phosphate buffers. The average values of tiyo determinations in 1 ,I2 \odium chloride, 0.02 p phosphate solution, pH 6.4 are: DA = 0.78 X lo-' cm.2 'sec. arid D , = 1.08 X lo-' cm.2,'sec. for water at 20°C. is the diffusion constant calculated by the method of moments. The diffusion curves obtained in 0.02 p phosphate buffer, pH 7.10, were found to more nearly approach the ideal Gaussian distrihution law, and are shown in (water a t 20"C.), figure 7. The yalues, I>* = 1.52 x lo-' and D M = 1.78 X which were obtained from these curves are too high, howeyer, because there
958 4270 7043 MIN. FIG. 7. Diffusion photographs ioi rabbit liver nucleoprotrin in 0.02 p phosphate buffer, p H 7.1.
with normal Gaubsian Fro. S. Coinpaiisc~nof esprrinicntal diffusion ('urvc jcirc~lt~s) 1ivc.r nuclwprotf~inin 0.02 p phosphate buffer, pH distribution curve (solid I i n ( ~ ~ Rxhbit . 7.1. Diffusion tinic,. io64 m i n .
\\as insufficient hiifier prcvnt t o repre- the tlifiii*ion potential -et u p by the rapidly diffusing gegenions of the nucleoprotein mdecule*.
c U,CTI, x n o v TIT-o qunntitie5 c ~ t nhe calculated from the wdimrntation and clitfu>ion constants reported above. They are ( 1 ) the moleciilar weight of the unhycirated molecule, and ( 2 ) tlie relati\-e degree of a-ymmetry of the molecule. The molecular weight i4 ohtainecl from the formula,
215
R-iBBIT LITER SUCLEOPROTEIN
ivhei-e s = 26.3 X lowJ3cm. 'sec./tlyne 'g. 1' = 0.658 C C . g. p = 0.9982 g. C C . I ) = 0.2-1.0 x 10-7 cm.? sec. R = 8.313 X 10' tlyne cm. degree T = 293'-1.
The iewlt is:
-If The frictional ratio, f
=
1,600,000-2,300,000
I,,, can be calcnlated b y means of
the equation: (6)
using the same constant3 a!: were used in equation 5. The result is:
flfo
=
2.6-3.3
By askuming that the nucleoprotein molecule has the shape of an elongated ellipsoitl, the Perrin equation can he applied:
where
0 a (axial ratio), b = equatorial radius, and a = 3 length of the axis of rotation
p =
The axial ratio calciilated for rabbit nucleoprotein i, then within the range of 1 '40 to I, 60. sUV\l iR'I
1. The method 01 X r s k y arid Pollister ha- heen i i d t o isolate nucleoprotein irom rabbit liver. 2. The presence and approkimate perventage of niic7lrir acid in rabbit Iir er nucleoprotein were determined by mean5 of it3 ultra\ iolet absorption spectrum and its phosphorus content. Per cent nucleic arid = 50. 3. The Dische diphenylaniine color reaction \\-a? I I - C ~ to demonstrate the pre*ence of desoxyribose nucleic acid in the rabbit liver niicleoprotein. 4. Electrophoretic mobility determination.: on rabbit lii er nucleoprotein in dilute aqiieoiis phoyphate buffers .hen- that it- mobilitie- approximate those obtained by Hall for calf thymu5 nuchhi5tone. 5 . 3fobilitiey of rabbit li\er nucleoprotein in 1 J I *odium chloride solution \\-ere found t o be 20-30 pcr (wit loner than in dilute acjueou~phosphate buffers. -MY,o,the nucleoprotein exhibited a tendency to dih-ociate into protein and niicleic acid in 1 -1I iodiiim chloride sollition..
G . Sedimentation, diffusion, aiid electrophoretic nieasurements indicate that rabbit l i n x nucleoprotc.iii, as prepnred by tlic method of llirsky and Pollister, i- inhomogeneous I\ it11 respect to i t h moleciilar Linetic and electrokinetic propcrtieu. 7 . Combined inea-uremt.iit- of ~cc1iniciit:itioii aiitl diffubion constant,- yield n molccular Jreight uhich range3 hetn-een 1,G00,000 and 2,300,000 and an a s i d ratio for the nuclroplotrin niolccult. ixhicli lie- 1)eturcw 40: 1 and 60: 1.
