BIOPOLYMERS
VOL. 16 (1977)
COMMUNICATIONS TO THE EDITOR Turbidimetric Determination o f Bacteriophage Molecular Weights
INTRODUCTION Turbidity measurements may be used to determine the molecular weights of high polymers,1,2including viru~es.’.~Thus far, turbidimetric studies have been reported only on viruses of molecular weights less than 50 X 106. We were interested in exploring whether the technique was reliable for larger particles. Therefore, we have used turbidimetry to determine the molecular weights of bacteriophages T2, T4, T5, and A, all of which have been reported”0 to have molecular weights in the range of 50-200 X lo6. T o determine molecular weight M , from turbidity T , we used the standard equation’
HQ lim
(c/T) =
1/M,
c--O
where
H =32~~n~(dn/dc)~/3NA~
(2)
In these equations Q is the particle dissipation factor;’ c, the concentration in g/ml; n, the refractive index; dnldc, the specific refractive index increment; N, Avogadro’s number; and A, the wavelength.
EXPERIMENTAL METHODS Phage concentrations were determined by measuring the absorbance at 260 nm. Extinction coefficients for T4 and T 5 are 13.36cm2/mg and 14.24 cm2/mg, re~pectively.~ T2 was assumed to have the same extinction coefficient as T4. The extinction coefficient for X was determined in our laboratory to be 13.34 cm2/mg. Q was interpolated from the tables of Doty and Steiner’ for spheres, since the heads of all of these viruses contribute most of the scattering, and they are roughly spherical. From electron microscopy, the head radii were taken as T2, T4: 500 As; T5: 450 A”; and A: 300 kg For light of 436 nm wavelength, these radii yie:d Q values of 0.701,0.742, and 0.874. An attempt was also made to determine Q from the equation (dlogrldlogh) = 4 - 6 for T4. Once TABLE I Molecular Weights (X of Bacterial Viruses Virus T2
Turbidimetry 249 f 20
T4
213 f 18
T5
119 f 9.5 59.9 f 3.9
X
Other 214 234 220 192.5 f 6 203 109.2 f 4 52 62 2797
0 1977 by John Wiley & Sons, Inc.
Ref. 4
5 6 7 8 7 9 10
BIOPOLYMERS VOL. 16 (1977)
2798
1 0.2
-0
0.12
0.5
,
0.24
0.30
-
0
0
Fig. 1.
0.18
C (mg/ml)
C/T vs c
T4
0.2
0.4 C ( mg/ml)
0.6
0.8
a t 436 nm for bacteriophages. A T 5 ( O ) ,T2 ( 0 ) . B h ( O ) , T4 (m).
@ is determined, Q can be obtained from the tables of Doty and Steiner.' The values of 6
obtained resulted in values for molecular weight and diameter about one-third smaller than accepted values. This is probably due to low turbidity at higher wavelengths,with consequent imprecision in the slope. Refractive indices were determined with a Brice-Phoenix differential refractometer. We measured dnlbc a t 436 nm to be 0.193 ml/g for h phage, and assumed it to be 0.190 ml/mg for T2, T4, and T5.3 Bacteriophage T2 and T4 were grown on E. coli Bl5 as described by Aksiyote-Benbasat and Bloomfield.12 T 5 was grown on E. coli F in nutrient broth (8 g nutrient broth, 5 g NaCl, 1.0 ml1M CaC12,ll. HzO) as described in Adams.13 E. coli MS 107 (C857S7), which contains h DNA in the prophage state, was grown on lambda broth (5 g NaCl, 10g tryptone, 2.5 g yeast extract, 1mg thiamine HCI, 1g maltose). Growth of h was induced by shaking 30 min at 42OC. The virus was purified by CsCl gradient ~entrifugati0n.l~Virus preparations were clarified for turbidity measurements by repeated differential centrifugation. Buffers were millipored to remove dust.
COMMUNICATIONS T O T H E EDITOR
2799
RESULTS Turbidity spectra were recorded from 700 to 350 nm, but only the value of the turbidity at 436 nm was used in the calculations. c l r vs c was plotted (see Fig. l ) , and extrapolated to c = 0 to determine molecular weight according to Q.(1). Results are given in Table I, where they are compared with experiments in the literature. Agreement is reasonable, within experimental error. I t appears from these results that molecular weights of large phage may be determined by turbidity with precision and accuracy better than lo%,so long as the dimensions are known from electron microscopy. This work was supported in part by NIH Grant GM 17855. References 1. Doty, P. & Steiner, R. F. (1950) J. Chem. Phys. 18,1211-1220. 2. Debye, P. (1947) J. Phys. Colloid Chem. 51,1€&31. 3. Camerini-Otero, R. D., Franklin, R. M. & Day, L. A. (1974) Biochemistry 13,37633773. 4. Cummings, D. J. & Kozloff, L. M. (1960) Biochim. Biophys. Acta 44,445-458. 5. Bendet, I. J., Swaby, L. G. & Lauffer, M. A. (1957) Biochim.Biophys. Acta 25,252262. 6 . Taylor, N. W., Epstein, H. T. & Lauffer, M. A. (1955) J. Am. Chem. SOC.77, 12701273. 7. Dubin, S. B., Benedek, G. B., Bancroft, F. C. & Freifelder, D. (1970) J . Mol. Biol. 54, 547-556. 8. Welch, J. B. (1976) Ph.D. thesis, University of Minnesota. 9. Dyson, R. D. & Van Holde, K. E. (1967) Virology 33,559-566. 10. Buchwald, M., Steed-Glaister, P. & Siminovitch, L. (1970) Virology 42,375-389. 11. Williams, R. C. & Fisher, H. W. (1974) An Electron Micrographic Atlas of Viruses, Thomas, Springfield, Ill., pp. 108-1 10. 12. Aksiyote-Benbasat, J. & Bloomfield, V. A. (1975) J. Mol. Biol. 95,336337. 13. Adams, M. H. (1959) Bacteriophage, Interscience, New York, 454-464. 14. Williams, P. H., Boyer, H. W. & Helinski, D. R. (1973) Proc. Natl. Acad. Sci. USA 70, 3744-3748.
D. M. BAHLS V. A. BLOOMFIELD Department of Biochemistry University of Minnesota St. Paul, Minnesota Received February 17,1977 Accepted April 11,1977
VOL. 16 (1977)
COMMUNICATIONS TO THE EDITOR Turbidimetric Determination o f Bacteriophage Molecular Weights
INTRODUCTION Turbidity measurements may be used to determine the molecular weights of high polymers,1,2including viru~es.’.~Thus far, turbidimetric studies have been reported only on viruses of molecular weights less than 50 X 106. We were interested in exploring whether the technique was reliable for larger particles. Therefore, we have used turbidimetry to determine the molecular weights of bacteriophages T2, T4, T5, and A, all of which have been reported”0 to have molecular weights in the range of 50-200 X lo6. T o determine molecular weight M , from turbidity T , we used the standard equation’
HQ lim
(c/T) =
1/M,
c--O
where
H =32~~n~(dn/dc)~/3NA~
(2)
In these equations Q is the particle dissipation factor;’ c, the concentration in g/ml; n, the refractive index; dnldc, the specific refractive index increment; N, Avogadro’s number; and A, the wavelength.
EXPERIMENTAL METHODS Phage concentrations were determined by measuring the absorbance at 260 nm. Extinction coefficients for T4 and T 5 are 13.36cm2/mg and 14.24 cm2/mg, re~pectively.~ T2 was assumed to have the same extinction coefficient as T4. The extinction coefficient for X was determined in our laboratory to be 13.34 cm2/mg. Q was interpolated from the tables of Doty and Steiner’ for spheres, since the heads of all of these viruses contribute most of the scattering, and they are roughly spherical. From electron microscopy, the head radii were taken as T2, T4: 500 As; T5: 450 A”; and A: 300 kg For light of 436 nm wavelength, these radii yie:d Q values of 0.701,0.742, and 0.874. An attempt was also made to determine Q from the equation (dlogrldlogh) = 4 - 6 for T4. Once TABLE I Molecular Weights (X of Bacterial Viruses Virus T2
Turbidimetry 249 f 20
T4
213 f 18
T5
119 f 9.5 59.9 f 3.9
X
Other 214 234 220 192.5 f 6 203 109.2 f 4 52 62 2797
0 1977 by John Wiley & Sons, Inc.
Ref. 4
5 6 7 8 7 9 10
BIOPOLYMERS VOL. 16 (1977)
2798
1 0.2
-0
0.12
0.5
,
0.24
0.30
-
0
0
Fig. 1.
0.18
C (mg/ml)
C/T vs c
T4
0.2
0.4 C ( mg/ml)
0.6
0.8
a t 436 nm for bacteriophages. A T 5 ( O ) ,T2 ( 0 ) . B h ( O ) , T4 (m).
@ is determined, Q can be obtained from the tables of Doty and Steiner.' The values of 6
obtained resulted in values for molecular weight and diameter about one-third smaller than accepted values. This is probably due to low turbidity at higher wavelengths,with consequent imprecision in the slope. Refractive indices were determined with a Brice-Phoenix differential refractometer. We measured dnlbc a t 436 nm to be 0.193 ml/g for h phage, and assumed it to be 0.190 ml/mg for T2, T4, and T5.3 Bacteriophage T2 and T4 were grown on E. coli Bl5 as described by Aksiyote-Benbasat and Bloomfield.12 T 5 was grown on E. coli F in nutrient broth (8 g nutrient broth, 5 g NaCl, 1.0 ml1M CaC12,ll. HzO) as described in Adams.13 E. coli MS 107 (C857S7), which contains h DNA in the prophage state, was grown on lambda broth (5 g NaCl, 10g tryptone, 2.5 g yeast extract, 1mg thiamine HCI, 1g maltose). Growth of h was induced by shaking 30 min at 42OC. The virus was purified by CsCl gradient ~entrifugati0n.l~Virus preparations were clarified for turbidity measurements by repeated differential centrifugation. Buffers were millipored to remove dust.
COMMUNICATIONS T O T H E EDITOR
2799
RESULTS Turbidity spectra were recorded from 700 to 350 nm, but only the value of the turbidity at 436 nm was used in the calculations. c l r vs c was plotted (see Fig. l ) , and extrapolated to c = 0 to determine molecular weight according to Q.(1). Results are given in Table I, where they are compared with experiments in the literature. Agreement is reasonable, within experimental error. I t appears from these results that molecular weights of large phage may be determined by turbidity with precision and accuracy better than lo%,so long as the dimensions are known from electron microscopy. This work was supported in part by NIH Grant GM 17855. References 1. Doty, P. & Steiner, R. F. (1950) J. Chem. Phys. 18,1211-1220. 2. Debye, P. (1947) J. Phys. Colloid Chem. 51,1€&31. 3. Camerini-Otero, R. D., Franklin, R. M. & Day, L. A. (1974) Biochemistry 13,37633773. 4. Cummings, D. J. & Kozloff, L. M. (1960) Biochim. Biophys. Acta 44,445-458. 5. Bendet, I. J., Swaby, L. G. & Lauffer, M. A. (1957) Biochim.Biophys. Acta 25,252262. 6 . Taylor, N. W., Epstein, H. T. & Lauffer, M. A. (1955) J. Am. Chem. SOC.77, 12701273. 7. Dubin, S. B., Benedek, G. B., Bancroft, F. C. & Freifelder, D. (1970) J . Mol. Biol. 54, 547-556. 8. Welch, J. B. (1976) Ph.D. thesis, University of Minnesota. 9. Dyson, R. D. & Van Holde, K. E. (1967) Virology 33,559-566. 10. Buchwald, M., Steed-Glaister, P. & Siminovitch, L. (1970) Virology 42,375-389. 11. Williams, R. C. & Fisher, H. W. (1974) An Electron Micrographic Atlas of Viruses, Thomas, Springfield, Ill., pp. 108-1 10. 12. Aksiyote-Benbasat, J. & Bloomfield, V. A. (1975) J. Mol. Biol. 95,336337. 13. Adams, M. H. (1959) Bacteriophage, Interscience, New York, 454-464. 14. Williams, P. H., Boyer, H. W. & Helinski, D. R. (1973) Proc. Natl. Acad. Sci. USA 70, 3744-3748.
D. M. BAHLS V. A. BLOOMFIELD Department of Biochemistry University of Minnesota St. Paul, Minnesota Received February 17,1977 Accepted April 11,1977
