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.

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(Recei ved 23 Sept ember 1968)

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