- Email: [email protected]
Quantitative estimation of bacteriochlorophyll in situ
COMMUNICATIONS M.E., Biochim. Biophys. rlcta 160, 204 (1968). 5. SAYRE, F. W., FISWLER, M. C., HUMPHREYS, G. K., AND JAYKO, M. E., Biochim. Biophys. Acta 160, 63 (1968). 6. HIEB, W. F., AND ROTHSTEIN, M., Science 160, 778 (1969). 7. BUECHER, E. J., HANSEN, E. AND YARWOOD, E. A., Proc. Sot. Exp. Biol. Med. 121, 390
(1966).
a FIG. 2. Growth curves for C. briggsae in basal medium supplemented with the following: autoclaved “Fraction A” plus autoclaved E. coli (O-O); autoclaved E. coli (A-A); autoclaved “Fraction A” (O-U). Nematodes were grown in 50-ml Erlenmeyer flasks containing 5.0 ml of basal medium (similar to the totally defined medium, but with soy-peptone in place of amino acids) (9), 0.46 mg of cholesterol in Tween 80 (6), approximately 4 mg (dry weight) of E. coli cells and Fraction A equivalent to 0.7 ml of the original heated liver extract from which the fraction was isolated. All components of the medium were autoclaved together before inoculation with nematodes. Worm counts and sterility checks were made as previously reported (9). protein of any special configuration by Sayre et al. (4, 5).
as reported
REFERENCES 1. DOUGHERTY, E. C., HANSEN, E. L., NICHOLAS, W. L., MOLLET, J. A., AND YARWOOD, E. A., Ann. N. Y. Acad. Sci. 77, 176 (1959). 2. NICHOLAS, W. L., DOUGHERTY, E. C., AND HANSEN, E. L., Ann. N. Y. Acad. Sci. 77,
218 (1959). 3. SAYRE, F. W., LEE, R. T., SANDMAN, R. P., AND PEREZ-MENDEZ, G., Arch. Biochem. Biophys. 118, 58 (1967). 4. SAYRE, F. W., FISHLER, M. C., AND JAYKO,
8. SAYRE, F. W., HANSEN, E. L., AND YARWOOD, E. A., Exp. Parasltol. 13, 98 (1963). 9. TOMLINSON, G. A., AND ROTHSTEIN, M., B&him. Biophys. Acta 63,465 (1962). 10. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). W. F. HIEB~ MORTON ROTHSTEIN Biology Department State University of New York at Buffalo Buj’alo, New York 14614 Received October 29, 1969; accepted November 31, 1969.
3 Present address: Department of Nutritional Sciences, University of California, Berkeley, California 94720
Quantitative
Estimation
of Bacteriochlorophyll
in Situ
The quantity of bacteriochlorophyll (BChl) in intact cells or subcellular particles from photosynthetic bacteria is frequently used as a basis of reference for comparing various photochemical and biochemical activities and as an index of the amount of energy-converting membrane present in cells growing under different conditions. Chlorophyll concentrationis ordinarily estimated from spectrophotometric measurements on organic solvent extracts (e. g., acetone-methanol) of cells, tissues, etc. With the bacteria, the most widely used procedure of this kind is the one described by Cohen-Basire et al. (1). One of the problems encountered with such methods is that the optical absorbancy (A) determination must be made as quickly as possible, because BChl is somewhat unstable in organic solvents (especially when exposed to light). This and the requirement for at least two time-consuming centrifugations are disadvantages for various kinds of experiments. Also, it is likely that BChl may not be quantitatively extracted by a single extraction from cells which contain relatively large quantities of the pigment. These
579
COMMUNICATIONS
400
500
I I I I I 600 700 800 Wavelength (nm)
I
I 900
FIG. 1. Absorption spectrum of Rhodopseudomonas cap&ala cells suspended in 25% bovine serum albumin. The cells were grown photosynthetically at saturating light intensity in a medium containing nn-malate, (NH&S04, thiamin, and mineral salts (3). Five milliliters of a 4-hr-old subculture (approximately 330 Mg dry weight/ml) were centrifuged, and the cells were resuspended in BSA as described in Procedure. The spectrum was obtained with a Zeiss PMQII spectrophotometer (l-cm light path cuvettes). considerations led us to develop a simple, rapid, and reliable procedure for making quantitative determinations of BChl in situ. The method is based on spectrophotometric measurements of intact bacteria, suspended in a medium the refractive index of which closely corresponds to that of the cells. As shown by Barer (2), under these conditions light-scattering due to gradients of refractive index between cells and medium is minimized, thereby yielding essentially clarified suspensions. Such “transparent suspensions” show sharp absorption spectra of intracellular pigments such as BChl (2). Preliminary tests with Rhodopseudomonas capsulata (American Type Culture Collection no. 23782) showed that minimal scattering is obtained with 25% bovine serum albumin (BSA; made by diluting 3O”r, BSA obtained from Sigma Chemical Co., St. Louis, MO. or Armour Pharmaceutical Co., Chicago, Ill.) as the supporting agent. The absorption spectrum of R. capsulata cells in this medium is given in Fig. 1. As compared to ordinary aqueous suspensions, ratios of peak-to-trough absorbancies (e. g., 860 nm relative to 660 nm) are high (cf. Fig. 1 in Ref. 1). This suggested that the light-scattering background in 25y0 serum albumin might not contribute greatly to the total absorbancy observed at the 860 nm. BChl peak. If this is so, over a cert.ain
range there should be a linear relation between A~Eo-A~~o and BChl concentration, determined by extraction of the pigment in the usual fashion. A relation of this kind is shown in Fig. 2, which can be used for quantitative estimation of BChl in intact cells. Procedure for R. capsulata. 5 ml of culture are centrifuged in a glass tube to sediment the bacteria. The supernatant fluid is decanted as completely as possible, and any droplets on the edge of the tube removed with absorbent paper. The centrifuge tube is vigorously vibrated on a Vortex, or similar, mixer in order to disperse the cells thoroughly in the small quantity of residual medium present. Three milliliters of 25y0 BSA are carefully added, the tube covered with Parafilm, and the cells uniformly suspended by gently inverting the tube (3 to 5 times). Absorbancies at 860 and 660 nm are measured, using 25’% BSA as the blank. The general applicability of the method, for culture suspensions containing 0.25-4 pg BChl/ml, is indicated by the fact that the 65 points defining the statistical function shown in Fig. 2 were obtained with cells of widely different BChl content (achieved by altering light intensity, oxygen tension, etc., during growth). For various reasons, the details of the method may require slight modifications for different applications. With Rhotbspirillum rubr?Lm (strain
COMMUNICATIONS
580
REFERENCES 1. COHEN-BAZIRE, G., SISTROM, W. R., AND STANIER, R. Y., J. Cell Camp. Physiot. 49.25 (1957).
2. BARER, R., Science 121, 709 (1955). 3. SOJKA, G. A., AND GEST, H., Proc. Nat. Acad. Sci. U.S.A. 61, 1486 (1968). 4. CLAYTON, R. K., Biochim. Biophys. Acta 76, 312 (1963). GARY A. SOJKA HUDSON H. FREEZE I I
pg
2
BChl/mI
3
4
of culture
FIG. 2. Relationship between in situ BChl absorbancy and BChl content of R. cupsulata cells. Absorbancies in 25yo BSA were determined as detailed in the text. BChl was estimated by the method of Cohen-Bazire et al. (1) on 5-ml culture samples; in each instance, the packed cells were resuspended in 0.1 ml of water, and the BChl was extracted with 4.9 ml of acetone:methanol (7:2, v/v). Absorbancy of the extract at 775 nm was measured (in cuvettes with l-cm light path), and BChl/ml of culture was calculated using an extinction coefficient of 75 rnM-‘cm-1 (4). The line was fitted by the method of least squares, based on 65 separate determinations (for the sake of clarity some coincident experimental points have been omitted).
Sl), the following changes were found to be necessary: 0.1 ml of distilled water must be added to the cell pellet to facilitate dispersal of the bacteria before addition of BSA; 3Oye BSA is used, rather than 25%; the Aa80-As60difference is measured. Twelve determinations with R. rubrum yielded a statistical calibrationline with a slope of 0.24 and a y-axis intercept of -0.025 (i.e., a slightly steeper line than with R. cupsulata). It should be noted that with R. rubrum the absolute absorbancy values tend to decrease slowly for about 30 min (at room temperature and ambient light) after resuspension in BSA; the AB~~-A~v, difference, however, remains constant. The procedure described takes less than 10 min for a determination and requires no special spectrophotometric accessories. We have found it to be particularly useful for studies on the kinetics of BChl synthesis. ACKNOWLEDGMENTS This investigation was supported by Grant GB-7333X from the National Science Foundation. We thank Donald E. Gest for technical assistance.
HOWARD GENT Deportment of Microbiology Indiana University Bloomington, Indiana 47401 Received November 7, 1969; accepted December 8, 1969
A Dissociable Reductase
Mutant from
Form of Dihydrofolate
Diplococcus
pneumonioe’
Dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NAD(P)-oxido-reductase, ED 1.6.1.3) from wild-type Diplococcus pneumoniae was shown (1, 2) by molecular-sieve chromatography to have a molecular weight (mol wt) of about 20,000. The same enzyme in a mutant strain (amel-3) exists in more than one form. Only a small fraction of the enzyme (approx. 10%) had elution properties corresponding to a molecular weight of 20,609, while the majority of the enzyme appeared to be considerably larger (1, 2). Although by no means conclusive, the results were best explained as an increase in molecular weight due to the association of the normal 20,000 mol wt unit in this strain. Other evidence lending further support to this idea is presented here. A more adequate chromatographic comparison of mutant and wild-type forms was possible with the newer Sepharose 4B (Pharmacia). In addition, partial dissociation of one of the two mutant forms now distinguishable was achieved by treatment with denaturants, urea, and guanidine hydrochloride. Isolation of the amel- strain of D. pneumoniue, cultural conditions and methods of genetic analysis, and the preparation of cell-free extracts have been described (3-7). Cells suspended in 0.05 M Tris + 0.001 M EDTA (pH 7.4) were disrupted by sonication and cellular debris removed by ceni The authors express appreciation to Doctor Dorris J. Hutchison for the interest offered during these studies. This work was supported in part by Grant CA 08748 from the National Cancer Institute and by Grant T-107 from the American Cancer Society.
(1966).
a FIG. 2. Growth curves for C. briggsae in basal medium supplemented with the following: autoclaved “Fraction A” plus autoclaved E. coli (O-O); autoclaved E. coli (A-A); autoclaved “Fraction A” (O-U). Nematodes were grown in 50-ml Erlenmeyer flasks containing 5.0 ml of basal medium (similar to the totally defined medium, but with soy-peptone in place of amino acids) (9), 0.46 mg of cholesterol in Tween 80 (6), approximately 4 mg (dry weight) of E. coli cells and Fraction A equivalent to 0.7 ml of the original heated liver extract from which the fraction was isolated. All components of the medium were autoclaved together before inoculation with nematodes. Worm counts and sterility checks were made as previously reported (9). protein of any special configuration by Sayre et al. (4, 5).
as reported
REFERENCES 1. DOUGHERTY, E. C., HANSEN, E. L., NICHOLAS, W. L., MOLLET, J. A., AND YARWOOD, E. A., Ann. N. Y. Acad. Sci. 77, 176 (1959). 2. NICHOLAS, W. L., DOUGHERTY, E. C., AND HANSEN, E. L., Ann. N. Y. Acad. Sci. 77,
218 (1959). 3. SAYRE, F. W., LEE, R. T., SANDMAN, R. P., AND PEREZ-MENDEZ, G., Arch. Biochem. Biophys. 118, 58 (1967). 4. SAYRE, F. W., FISHLER, M. C., AND JAYKO,
8. SAYRE, F. W., HANSEN, E. L., AND YARWOOD, E. A., Exp. Parasltol. 13, 98 (1963). 9. TOMLINSON, G. A., AND ROTHSTEIN, M., B&him. Biophys. Acta 63,465 (1962). 10. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). W. F. HIEB~ MORTON ROTHSTEIN Biology Department State University of New York at Buffalo Buj’alo, New York 14614 Received October 29, 1969; accepted November 31, 1969.
3 Present address: Department of Nutritional Sciences, University of California, Berkeley, California 94720
Quantitative
Estimation
of Bacteriochlorophyll
in Situ
The quantity of bacteriochlorophyll (BChl) in intact cells or subcellular particles from photosynthetic bacteria is frequently used as a basis of reference for comparing various photochemical and biochemical activities and as an index of the amount of energy-converting membrane present in cells growing under different conditions. Chlorophyll concentrationis ordinarily estimated from spectrophotometric measurements on organic solvent extracts (e. g., acetone-methanol) of cells, tissues, etc. With the bacteria, the most widely used procedure of this kind is the one described by Cohen-Basire et al. (1). One of the problems encountered with such methods is that the optical absorbancy (A) determination must be made as quickly as possible, because BChl is somewhat unstable in organic solvents (especially when exposed to light). This and the requirement for at least two time-consuming centrifugations are disadvantages for various kinds of experiments. Also, it is likely that BChl may not be quantitatively extracted by a single extraction from cells which contain relatively large quantities of the pigment. These
579
COMMUNICATIONS
400
500
I I I I I 600 700 800 Wavelength (nm)
I
I 900
FIG. 1. Absorption spectrum of Rhodopseudomonas cap&ala cells suspended in 25% bovine serum albumin. The cells were grown photosynthetically at saturating light intensity in a medium containing nn-malate, (NH&S04, thiamin, and mineral salts (3). Five milliliters of a 4-hr-old subculture (approximately 330 Mg dry weight/ml) were centrifuged, and the cells were resuspended in BSA as described in Procedure. The spectrum was obtained with a Zeiss PMQII spectrophotometer (l-cm light path cuvettes). considerations led us to develop a simple, rapid, and reliable procedure for making quantitative determinations of BChl in situ. The method is based on spectrophotometric measurements of intact bacteria, suspended in a medium the refractive index of which closely corresponds to that of the cells. As shown by Barer (2), under these conditions light-scattering due to gradients of refractive index between cells and medium is minimized, thereby yielding essentially clarified suspensions. Such “transparent suspensions” show sharp absorption spectra of intracellular pigments such as BChl (2). Preliminary tests with Rhodopseudomonas capsulata (American Type Culture Collection no. 23782) showed that minimal scattering is obtained with 25% bovine serum albumin (BSA; made by diluting 3O”r, BSA obtained from Sigma Chemical Co., St. Louis, MO. or Armour Pharmaceutical Co., Chicago, Ill.) as the supporting agent. The absorption spectrum of R. capsulata cells in this medium is given in Fig. 1. As compared to ordinary aqueous suspensions, ratios of peak-to-trough absorbancies (e. g., 860 nm relative to 660 nm) are high (cf. Fig. 1 in Ref. 1). This suggested that the light-scattering background in 25y0 serum albumin might not contribute greatly to the total absorbancy observed at the 860 nm. BChl peak. If this is so, over a cert.ain
range there should be a linear relation between A~Eo-A~~o and BChl concentration, determined by extraction of the pigment in the usual fashion. A relation of this kind is shown in Fig. 2, which can be used for quantitative estimation of BChl in intact cells. Procedure for R. capsulata. 5 ml of culture are centrifuged in a glass tube to sediment the bacteria. The supernatant fluid is decanted as completely as possible, and any droplets on the edge of the tube removed with absorbent paper. The centrifuge tube is vigorously vibrated on a Vortex, or similar, mixer in order to disperse the cells thoroughly in the small quantity of residual medium present. Three milliliters of 25y0 BSA are carefully added, the tube covered with Parafilm, and the cells uniformly suspended by gently inverting the tube (3 to 5 times). Absorbancies at 860 and 660 nm are measured, using 25’% BSA as the blank. The general applicability of the method, for culture suspensions containing 0.25-4 pg BChl/ml, is indicated by the fact that the 65 points defining the statistical function shown in Fig. 2 were obtained with cells of widely different BChl content (achieved by altering light intensity, oxygen tension, etc., during growth). For various reasons, the details of the method may require slight modifications for different applications. With Rhotbspirillum rubr?Lm (strain
COMMUNICATIONS
580
REFERENCES 1. COHEN-BAZIRE, G., SISTROM, W. R., AND STANIER, R. Y., J. Cell Camp. Physiot. 49.25 (1957).
2. BARER, R., Science 121, 709 (1955). 3. SOJKA, G. A., AND GEST, H., Proc. Nat. Acad. Sci. U.S.A. 61, 1486 (1968). 4. CLAYTON, R. K., Biochim. Biophys. Acta 76, 312 (1963). GARY A. SOJKA HUDSON H. FREEZE I I
pg
2
BChl/mI
3
4
of culture
FIG. 2. Relationship between in situ BChl absorbancy and BChl content of R. cupsulata cells. Absorbancies in 25yo BSA were determined as detailed in the text. BChl was estimated by the method of Cohen-Bazire et al. (1) on 5-ml culture samples; in each instance, the packed cells were resuspended in 0.1 ml of water, and the BChl was extracted with 4.9 ml of acetone:methanol (7:2, v/v). Absorbancy of the extract at 775 nm was measured (in cuvettes with l-cm light path), and BChl/ml of culture was calculated using an extinction coefficient of 75 rnM-‘cm-1 (4). The line was fitted by the method of least squares, based on 65 separate determinations (for the sake of clarity some coincident experimental points have been omitted).
Sl), the following changes were found to be necessary: 0.1 ml of distilled water must be added to the cell pellet to facilitate dispersal of the bacteria before addition of BSA; 3Oye BSA is used, rather than 25%; the Aa80-As60difference is measured. Twelve determinations with R. rubrum yielded a statistical calibrationline with a slope of 0.24 and a y-axis intercept of -0.025 (i.e., a slightly steeper line than with R. cupsulata). It should be noted that with R. rubrum the absolute absorbancy values tend to decrease slowly for about 30 min (at room temperature and ambient light) after resuspension in BSA; the AB~~-A~v, difference, however, remains constant. The procedure described takes less than 10 min for a determination and requires no special spectrophotometric accessories. We have found it to be particularly useful for studies on the kinetics of BChl synthesis. ACKNOWLEDGMENTS This investigation was supported by Grant GB-7333X from the National Science Foundation. We thank Donald E. Gest for technical assistance.
HOWARD GENT Deportment of Microbiology Indiana University Bloomington, Indiana 47401 Received November 7, 1969; accepted December 8, 1969
A Dissociable Reductase
Mutant from
Form of Dihydrofolate
Diplococcus
pneumonioe’
Dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NAD(P)-oxido-reductase, ED 1.6.1.3) from wild-type Diplococcus pneumoniae was shown (1, 2) by molecular-sieve chromatography to have a molecular weight (mol wt) of about 20,000. The same enzyme in a mutant strain (amel-3) exists in more than one form. Only a small fraction of the enzyme (approx. 10%) had elution properties corresponding to a molecular weight of 20,609, while the majority of the enzyme appeared to be considerably larger (1, 2). Although by no means conclusive, the results were best explained as an increase in molecular weight due to the association of the normal 20,000 mol wt unit in this strain. Other evidence lending further support to this idea is presented here. A more adequate chromatographic comparison of mutant and wild-type forms was possible with the newer Sepharose 4B (Pharmacia). In addition, partial dissociation of one of the two mutant forms now distinguishable was achieved by treatment with denaturants, urea, and guanidine hydrochloride. Isolation of the amel- strain of D. pneumoniue, cultural conditions and methods of genetic analysis, and the preparation of cell-free extracts have been described (3-7). Cells suspended in 0.05 M Tris + 0.001 M EDTA (pH 7.4) were disrupted by sonication and cellular debris removed by ceni The authors express appreciation to Doctor Dorris J. Hutchison for the interest offered during these studies. This work was supported in part by Grant CA 08748 from the National Cancer Institute and by Grant T-107 from the American Cancer Society.