Relationship Between the Sedimentation Coefficient and Molecular Weight of Bacteriophages

opossumoozeMechanics

Feb 21, 2014 (3 years and 8 months ago)

76 views

J. gen. Virol. (1969), 4, 577-583
Printed in Great Britain
577
Relationship Between the Sedimentation Coefficient
and Molecular Weight of Bacteriophages
By M. J. PI TOUT
National Nutrition Research Institute of the Council for Scientific and
Industrial Research, Pretoria, South Africa
J. D. CONRADI E AND A. J. VAN RENSBURG
Departments of Chemical Pathology and Microbiology,
University of Pretoria, Pretoria
(Accepted 2 December 1968)
SUMMARY
Bacteriophage PL25 was purified by centrifugation in caesium chloride.
It has S~°0,w = 485; D~o,w = o-68x IO -7 cm.~Jsec, and M.W. = 54"3x IOa.
An equation was derived relating S~0,w and molecular weight of bacterio-
phages.
I NTRODUCTI ON
The molecular weight of bacteriophages can be derived from light-scattering (Schito,
Rialdi & Pesce, 1966; Strauss & Sinsheimer, I963), sedimentation-diffusion (Mrller,
1964; Schito et al. 1966; Cummings & Kozloff, 196o; Davison & Freifelder, 1962;
Swanby, 1959; Goldwasser & Putnam, I952), sedimentation-viscosity (Schito et al.
1966) and sedimentation-equilibrium experiments (Dyson & van Holde, 1967), but deter-
minations are cumbersome. Different equations relating S~0,w and molecular weight
of DNA were described by Josse & Eigner (1966). Since similar equations for bacterio-
phages would be useful we decided to investigate physical properties of phage PL 25
with this in mind.
METHODS
Media. Difco brain-heart-infusion broth was used. Other media were described by
Coetzee & Sacks (196o). Incubation temperature was 37 °.
Phages and hosts. Phage PL25 and its host strain Providence NCTC 92I I (Coetzee,
1963) were used.
Preparation and purification of phage PL25. Phage lysates (5 x i on p.f.u./ml.) were
prepared by an agar-layer method (Coetzee & Sacks, 196o). Phage stocks were
sterilized by addition of o" I vol. chloroform. Crude lysates were purified by centrifuga-
tion at 8ooog for 3o min. at IO ° to remove agar. Supernatant fluids were then centri-
fuged at 3o,ooog for IOO min. at IO °, and the pellet suspended in o.I M-phosphate
buffer, pH 6.8, or o'15 M-NaCl+o.oI5 M-citrate buffer, pH 7"o, to a titre of about
5 x io 1~ p.f.u./ml. Initially the phage was purified by subjecting it to charcoal (o'079 g.
activated charcoal/ml, phage suspension), pronase (I/zg./ml. at pH 9"o) and combined
charcoal + pronase treatments. These treatments were discarded on account of unsatis-
37 J. Virol. 4
578 M.J. PITOUT, J. D. CONRADIE AND A. J. VAN RENSBURG
factory results. Therefore phage was finally purified by CsC1 density gradient centri-
fugation. A phage suspension of ~'5 ml. was layered on to 3"5 ml. CsC1 solution
( I 2"2 g. CsC1 in I4"4 ml. o.I M-phosphate buffer, pH 6.8) and centrifuged for I4 to
I6 hr at I8,ooog in a Spinco SW5o swinging bucket rotor. The phage band was
removed with a syringe and again centrifuged. Ten-drop fractions were collected from
the bottom of the tubes after puncture with a 22-gauge needle. The absorbence at
26o nm. of the fractions was measured in a Beckman DK-2A spectrophotometer using
I mm. cells. Fractions with an absorption above o'5 were collected and dialysed for
24 hr against five changes of o.r M-phosphate buffer, pH 6.8. The phage suspension
was then assayed for infectivity.
Estimation of DNA content of phage PL25. This was determined according to the
diphenylamine reaction (Kupila, Bryan & Stern, I96 0.
Determination of sedimentation coefficient of phage PL 25. Sedimentation coefficients
of various concentrations of phage PL25 were determined in the Spinco model E
ultracentrifuge equipped with ultraviolet optics. Relative concentrations of phage
suspensions were expressed in absorbency units at 260 nm. and determinations were
made between o.2 and I"5 units. The An-D and An-E rotors with I2 and 3o mm. cells
were used. Phage suspensions were centrifuged at 2o ° and 9945 rev./min. Photographs
were taken at 2 min. intervals on Kodak commercial film. Boundary positions were
determined by scanning photographs with a Beckman Analytrol densitometer. All
sedimentation coefficients were corrected to S~,w and the limit sedimentation coeffi-
S o o
cient (s0,w) was obtained by plotting Sa0,w values against concentration of phage,
followed by extrapolating to zero concentration.
Determination of diffusion coefficient of phage PL25. Boundary spreading in the
ultracentrifuge cell was analysed in terms of a true diffusion coefficient (M/Slier, I964).
Diffusion coefficients were calculated from
D = ~( I - St °2t ) C~ I [ 2 f ~
4 y2t , Co=2 I - ~ e-~" dy .
In these equations C, is the concentration at a distance z from the boundary; Co the
initial concentration; ~ the mean distance in cm. at a time t from a level in the boundary
where the concentration ratio (C/Co) is o'5 to the equidistant levels with concentration
ratios (C/Co) of o.2 and o.8 respectively. The factor y may be obtained from tables
giving the numeral values for the well-known probability integral:
(y) = ~ e-"dy = ! Co'
for which values are given for definite values of C/Co (Svedberg & Pederson, I94O).
Diffusion-coefficient experiments were performed at 5 ° using the An-E rotor with
12 and 30 mm. analytical cells. The rotor was spun at 9945 rev./min, for 3 min. to
establish a permanent plateau. When the boundary had moved about 0. 3 ram. from
the meniscus, the rotor was decelerated to the preset low-speed value of 2o95 rev./min.
Diffusion coefficients were determined at the same concentrations as sedimentation
coefficients. Exposure intervals were 64 min. All diffusion coefficients were corrected
to D~o,w and plotted against phage concentration and extrapolated to zero concentration
to give D~0,w.
Determination of partial specific volume. The density and partial specific volume @)
measurements were pycnometrically determined at 2o.o ° with the same solvent used
Molecular weights of bacteriophages 579
in the sedimentation-diffusion measurements. The value was calculated according to
Schachman (I957).
Determination of molecular weight of phage PL25. After standardization of the
sedimentation and diffusion coefficients to the same solvent (water) and temperature
(20 °) (Svedberg & Pedersen, 194o), the molecular weight of phage PL 25 was calculated
from the Svedberg equation (Svedberg & Pedersen, I94o)
RTs
M=
D(I -~p)
in which R=gas constant, 8.314×~o T erg/mol./degree; T=293°K; s=S~o,w see.;
D = D ° ao,w cm.Z/sec.; ~=partial specific vol. cm.a/g.; p= density of water at 293 °K.
0.6
: 0.4
,.Q
,<
0.2
240 260 280 300 320 340 360
Wavelength (nm)
Fig. I
03
/
)
Radial distance
Fig. 2
(c)
Fig. t. Ultraviolet absorption spectra of phage PL25 during purification. Curves a to d
represent the ultraviolet spectra after CsC1 density gradient centrifugation, charcoal treat-
ment, pronase treatment and a combined charcoal + pronase treatment respectively.
Fig. 2. Densitometer tracings of ultraviolet absorption photographs. Curves a to c represent
ultraviolet absorption photographs after treatment with pronase and charcoal, charcoal
and CsCI density gradient centrifugation respectively. Curves a and b indicate the presence
of low-molecular-weight ultraviolet absorbing material.
RESULTS
Purification of phage PL 2 5
Only CsC1 density gradient centrifugations yielded homogeneous preparations
(Fig. I, 2). It was necessary to repeat the CsC1 centrifugation to remove all impurities.
Purified phage sedimented as a single boundary indicative of homogeneous macro-
37-2
580 M.J. PI TOUT, J. D. CONRADI E AND A. J. VAN RENSBURG
molecular material. In addition, the plateau region (solvent) did not contain any
ultraviolet absorbing material (Fig. 2). The phage concentrations at various steps
during purification are presented in Table I.
Tabl e I. Purification of phage PL z5
Volume P.f.u. recovered
Sample (ml.) P.f.u./mL Total p.f.u. (%)
Crude lysate 2oo 3"0 x lO ix 6.0 x IO la ioo
Differential centrifugation 4"5 9"o X 10 TM 4"10 × I O TM 68
First CsC1 density gradient I-8 I-O x lO TM 1-80 x IO TM 30
centrifugation
Second CsC1 density gradient 0"7 2"3 x 1o TM 1.60 x 1o TM 27
centrifugation
Physical properties of phage PL 25
Sedimentation coefficients of the phage were determined from velocity sedimentation
runs at different concentrations. The sedimentation coefficient was independent of
concentration and an average value of 485 was obtained for S~0,w. Schito et aL (I966)
found the sedimentation coefficient of N4 coliphage to be concentration-dependent.
30
×
~' 20
I
×
10
o I I I I
1-0 2.0 3.0 4.0
t x lO 4 (see.)
Fig. 3. A representative plot of t ~( I - sw2t ) against t for the 2o (8o%) C/Co ratio in the
boundary, where ~ denotes the mean distance in cm. at a time t from a level in the boundary
where the concentration ratio C/C0 is o'5 to the equidistant levels with concentration ratios
(C/Co) of o.2 and o.8 respectively. The slope of the line, t an ~ = ti2(I --s~o~t)/t, corresponding
to the 2o(8o) point, was used for the calculation of the diffusion coefficient.
Spreading measurements made before, during, and after deceleration showed that, in
the absence of external braking, no deterioration of the boundary took place during
diffusion experiments. The diffusion coefficient was calculated by plotting u2(i-s~o~t)
against time (Fig. 3), where D=~( r -soj2t)/4y~t and y2= 1.4i 7 (Svedberg & Pedersen,
I94o).
Molecular weights of bacteriophages 58 I
The diffusion coefficient of phage PL25 was also independent of concentration and
an average value of o.68 x lO -7 cm.2/sec, was obtained. A value of o.68 ml./g, was
calculated for the partial specific volume (~), corresponding to a DNA content of 47 ~o
of the phage particle weight (Schito et al. 1966). The diphenylamine assay method
yielded a value of 48 %.
Table 2. Relationship between S~0,w and molecular weight of bacteriophages
Molecular weight
Phage S~°0,w x 1o 6 Reference
T6 lO5O I45"o Goldwasser & Putnam (1952)
T2 Io66 220.0 Taylor, Epstein & Lauffer (I955)
N4 615 83"0 Schito, Rialdi & Pesce (I966)
T 3 476 49"o Swanby (1959)
MS 2 8I-5 5"3 MOiler 0964)
CX- 174 I 14"o 6.2 Sinsheimer (1959)
T 7 487 38"0 Davison & Freifelder (I962)
TP-84 436 50"0 Saunders & Campbell (I966)
Lambda 416 57"0 Dyson & Van Holde (1967)
PL 25 485 54"0 This investigation
T 2 (fast form) I o 17 214"o Cummings & Kozloff (I 96o)
(slow form) 71o 216.o
Determination of molecular weight of bacteriophages
The substitution of the various values obtained in the Svedberg equation yields a
value of 54"3 x lO s for the molecular weight of phage PL 25.
The molecular weight of phage PL25 was correlated with known molecular weight
values of other bacteriophages (Table 2). A linear relationship was found between
S~o,w and the molecular weight of phage (on logarithmic scale). An empirical equation
relating S~0,w and molecular weight was derived:
S~o,w = I'II4X IO- aXM °'7~9
DISCUSSION
The procedures of Van Holde (I96O) and Mommaerts & Aldrich (I958) for deter-
mining the diffusion coeffÉcient of phage PL25 gave unsatisfactory results. With the
use of the Van Holde method (196o) the height/area value could not be determined
accurately. No boundary could be obtained with the synthetic boundary cell in the
Mommaerts & Aldrich (I958) procedure. This was probably due to sedimentation of
phage before layering of buffer could take place. The method of Chervenka (I966)
was attempted but was unsuccessful as sedimentation of the phage occurred at the
lowest possible rev./min, setting of the centrifuge. The discrepancy in the diffusion
coefficient with ultraviolet absorption optics (Fig. 3) was probably due to accumulation
of phage at the bottom of the cell.
Results presented in Table 2 and Fig. 4 indicate that only one phage molecular
weight deviates from the linear relationship with S~0,w. This value is for the slow form
of phage T2 (Cummings & Kozloff, I96O). Electron micrographs obtained by different
procedures show that the slow form has a longer head than the fast form. This abnorm-
ality is probably the cause of the anomalous diffusion coefficient and molecular weight
of the slow form ofT2. This form is obtained at a pH value of 5"7, while sedimentation
coefficients are normally measured at about neutral pH.
582 M.J. PITOUT, J. D. CONRADIE AND A. J. VAN RENSBURG
An empirical equation for the determination of molecular weights of spherical
RNA phages has been derived by Marvin & Hoffmann-Beding (I963). In this equation
M= I I5O ~S~i ~e~ ~
where S~0,w is the limit sedimentation coefficient; ~ the partial specific volume and
the density of the solution. Substituting the values of 485 and o.68 for S~0,w and
respectively in the Marvin & Hoffmann-Berling equation, an M value of 56"o x lO s
is obtained for phage PL25, which is in close agreement with the value procured from
our equation.
lO0( .f o ~ e~
o~ loc
I I I IIIIiJ i i i i i i111 I
lO 100
Molecular weight x io 6
Fig. 4. Sedimentation coefficient as a function of molecular weight. Bacteriophage MS 2,
~bX-174, T7, T3, TP-84, lambda, PL25, N4, T6 and T2 are represented by O, 0, D, III, A,
A, T, V, ~, and G, respectively. References to these phages are listed in Table 2.
The physical characterization of macromolecules assists in clarifying their biological
roles. This might also apply to bacteriophages. Our results indicate that molecular
weights of phages can be determined from a knowledge of their sedimentation
coefficients.
REFERENCES
CH~RVENKA, C. H. (1966). Use of combined schlieren and interference optics for the determination
of molecular weights from sedimentation equilibrium data. Analyt. Chem. 38, 356.
COEXZEE, J. N. (1963). Lysogeny in Providence strains and the host-range of Providence bacterio-
phages. Nature, Lond. x97, 515 .
CO~TZEE, J. N. & SACKS, T. G. (I96O). Transduction of streptomycin resistance in Proteus mirabilis.
J. gen. Microbiol. 23, 445.
CUMMINGS, D. J. & KozLorr, L. M. (196o). Biophysical properties of bacteriophages T 2. Biochim.
biophys. Acta 44, 445.
DAVlSON, P.F. & FREIFELDER, D. (I962). The physical properties of T7 bacteriophage. J. molec.
Biol. 5, 635.
Molecular weights of bacteriophages 583
DYSON, R. D. & VAN HOLDE, K. E. (I967). An investigation of Bacteriophage lambda, its protein
ghosts and subunits. Virology 33, 559.
GOLDWASSER, E. & PUTNAM, F. 0952). Physicochemical properties of T6 bacteriophage. J. bioL
Chem. x9o, 61.
JOSSE, J. & EIGNER, J. (I966). Physical properties of deoxyribonucleic acid. A. Rev. Biochem. 35, 789.
KUPmA, S., BRYAN, A. M. & STERN, H. (196I). Extractability of DNA and its determination in
tissues of higher plants. Plant Physiol. 36, 212.
MARVIN, D. A. & HOFFMANN-BERLING, H. (1963). A fibrous DNA phage (fd) and a spherical phage
(fr) specific for male strains of E. coil Z. Naturforsch. x8b, 884.
M6LLER, W. J. (I964). Determination of diffusion coefficients and molecular weights of ribonucleic
acids and viruses. Proc. natn. Acad. Sci. U.S.A. 5x, 5OL
MOMMAERTS, W. F. H. M. & ALDRICH, B.B. 0958). Determination of the molecular weight of
Myosin. Interference-optical measurements during the approach to ultracentrifugal sedimenta-
tion and diffusion equilibrium. Biochim. biophys. Acta ~8, 627.
SAUNDERS, G.F. & CAMPBELL, L.L. (I966). Characterization of thermophilic bacteriophage for
Bacillus stearothermophilus. J. Bact. 9 x, 340.
SCHACrtMAN, H. K. (t957)- Ultracentrifugation, diffusion and viscometry. Methods in Enzymology,
vol. LV, 32. Eds. S. P. Colowick and N. O. Kaplan. New York: Academic Press.
SCHITO, G.C., RtALDI, G. & PESCE, A. (I966). Biophysical properties of N4 coliphage. Biochim.
biophys. Acta x~'9, 482.
SrNSHEIMER, R. L. 0959)- Purification and properties of bacteriophages x-174. J. molec. Biol. x, 37.
STRAUSS, J. H. & StNSHEI~R, R. L. (I963). Purifications and properties of bacteriophage MS 2 and
of its ribonucleic acid. J. molec. BioL 7, 43.
SWDBERG, T. & PEDERSEN, K. O. 0940). The Ultracentrifuge. Oxford University Press.
SWANBY, L.S. 0959). Some biophysical properties of the T3 bacteriophage of Escherichia coll.
Diss. Abstr. zo, t567.
TAYLOR, N. W., EPSTEIN, H. T. & LAUFFER, M. A. (I955). Particle weight, hydration and shape of
the T z bacteriophage of Escherichia coli. J. Am. chem. Soc. 77, 127o.
VAN HOLDE, K. E. 0960). A modification of Fujita's method for the calculation of diffusion co-
efficients from boundary spreading in the ultracentrifuge. J. phys. Chem., Ithaca. 64, x582.
(Recei ved 23 Sept ember 1968)