INFECTION AND IMMUNrrY, Feb. 1978, p. 749-751
Vol. 19, No. 2
0019-9567/78/0019-0749$02.00/0 Copyright X 1978 American Society for Microbiology
Printed in U.S.A.
Acid Precipitation of Clostridium botulinum Type C and D Toxins from Whole Culture by Addition of Ribonucleic Acid as a Precipitation Aid MASAKAZU IWASAKI AND GENJI SAKAGUCHI* Department of Veterinary Science, College of Agriculture, University of Osaka Prefecture, Sakai-shi, Osaka
591, Japan Received for publication 15 September 1977
The ratios of ribonucleic acid to protein contents of Clostridium botulinum type C, D, and E cultures were lower than those of type A, B, and F cultures. Addition of ribonucleic acid at 0.4 mg/ml to culture satisfactorily aided acid precipitation of type C and D toxins, but not that of type E toxin. C. botulinum type A toxin has been obtained in crystalline form (1, 9). Type A, B, and F toxins have been purified by similar procedures in this laboratory (8, 12, 14). In purification of these toxins, acid precipitation was used as the first step to concentrate the toxin from whole culture. This procedure is relatively simple, safe, and inexpensive to perform when handling a large volume of culture. Type C, D, and E toxins have also been purified by investigators in this and other laboratories (3-5, 7, 15), but acid precipitation was not applied because of the poor recovery of toxin (3, 5). It is known that type A, B, and F toxins themselves are soluble at pH 4.0, but are precipitable at this pH only when they are in crude form. After treating the acid precipitate with protamine or ribonuclease, the toxin became soluble at pH 4.0 (8, 12, 14). Nucleic acids or some other acidic substances must have played an important role in the acid precipitation of the toxin (13). We attempted, therefore, to determine the ribonucleic acid (RNA) contents of cultures of types A through F and to add RNA to type C, D, and E cultures that contained less RNA than type A, B, and F cultures to see if it would aid acid precipitation of the toxins. Each strain of C. botulinum type A through F, the names of which are given in Table 1, was grown in a medium consisting of 2% peptone, 0.5% yeast extract, 0.5% glucose, and 0.1% Lcysteine-hydrochloride, pH 7.2, for 3 days at
300C. Protein contents were determined according to Lowry et al. (10) on the residue of cold and then hot 5% trichloroacetic acid extraction. RNA contents were determined according to Kerr and Seraidarian (6) on the cold trichloroacetic acid precipitate. Protein and RNA contents of cultures of type A through F are shown in Table 1. The ratio of
RNA to protein contents of type C, D, and E cultures were apparently lower than those of type A, B, and F cultures. We presumed that the toxin would be precipitated by acidification if the RNA-to-protein ratio of culture was made to 0.77 or higher. RNA (Yeast RNA, P-L Biochemical Inc., Milwaukee, Wis.) was dissolved in 2 N NaOH. The RNA solution was adjusted to pH 6.0 with 1 N HCI and brought to a final concentration of 10 mg/ml by the addition of distilled water. The cultures of types C, D, and E were each divided into two portions. To a portion of each culture, the RNA solution was added to a concentration of 0.4 mg/ml. Both portions were adjusted to pH 4.0 with 3 N H2SO4. The acidified cultures were allowed to stand for 3 h at room temperature. The precipitate formed was collected by centrifugation for 5 min at 1,500 x g and 4°C and resuspended in 0.2 M phosphate buffer, pH 6.0, to the original volume. Toxin was titrated by the time-to-death method by intravenous injection of a fourfold dilution into mice (2). Type B, E, and D (strain CB16 only) toxins were activated with trypsin before injection (7, 8, 11). The toxicities of acid precipitate obtained in the presence and absence of RNA are shown in Table 2. Without the aid of RNA, the recovery of the toxin was not satisfactory. In the presence of RNA at 0.4 mg/ml, the recovery of the toxin was satisfactorily high with all type C and D strains. Little or no toxin remained in the supernatant. The recovery of toxin was not satisfactory when RNA was added to 0.2 mg/ml or lower concentrations. With type E culture, acidification in the absence of RNA recovered 62% of the toxin in the centrifugal precipitate. This was due apparently to the association of type E toxin with the bacterial cells (7). Addition of RNA did not increase 749
INFECT. IMMUN.
NOTES
750
Type C C
TABLE 1. Protein and RNA contents of whole cultures of types A through F RNA (pg/ml) RNA/protein ratio Strain Protein (pg/ml) CB19 203
630 681
126 115
0.20 0.17
Stockholm
353
35
0.10
1873 CB16 4947
630 382 658
85 40 245
0.13 0.10 0.37
E
German sprats
438
244
0.55
A B B F
62
345
377
1.09
Okra
337
460
1.36
QC
388
346
0.89
Langeland
332
254
0.77
C D
D D
TABLE 2. Acid precipitation of type C, D, and E toxins with or without added RNA
LDgia/ml X 10-4 in: b Acid precipitate
Strain
Type
Whole culture RNA not added (%)
a
C C
CB19 203
C D D
Stockholm 1873 CB16
D
4947
E B
German sprats Okra
RNA added (%)
(35) (50)
19 35 170
22
(45) (17) 6.9 (31)
16
7.3 (46)
15
26 36 0.77 200
9.1 18 0.35 34
5.6 (62) 220 (92)
9.0 240
(73) (97)
0.64 (83) (85) 18 (82) (94)
6.0 (67) NDC
LD5o, 50% lethal dose.
b Numbers indicate the absolute amounts of toxin recovered. c
ND, Not done.
the recovery of the toxin in the precipitate. Type E toxin is adsorbed onto CM-Sephadex at pH 6.0 (7), whereas type B toxin is not under the same conditions (8). Type E toxin released from the bacterial cells, therefore, may differ in electrostatic property from toxins of other types. The extract of the acid precipitate of type C and D toxins obtained by the aid of RNA contained a large quantity of RNA, but the RNA was removed subsequently by the protamine treatment (8, 12, 14) without appreciable loss of the toxin. We have succeeded in purifying type C and D (11) toxins by applying the same subsequent procedures, namely chromatography on SP-Sephadex C-50 and molecular sieving on Sephadex G-200, as used for purifying type A, B, and F toxins (8, 12, 14). Thus, acid precipitation of type C and D toxins from whole culture in the presence of RNA added as a precipitation aid is very effective as the initial step of purification from large quantities of type C and D cultures. LITERATURE CITED 1. Abrams, A., G. Kegeles, and G. A. Hottle. 1946. The purification of toxin from Clostridium botulinum type A. J. Biol. Chem. 164:63-79.
2. Boroff, D. A., and U. Fleck. 1966. A statistical analysis of rapid in vivo method for the titration of the toxin of Clostridium botulinum. J. Bacteriol. 92:1580-1581. 3. Cardelia, M. A., J. T. Duff, C. Gottfried, and J. S. Begel. 1958. Studies on immunity to toxins of Clostridium botulinum. IV. Production and purification of type C toxin for conversion to toxoid. J. Bacteriol.
75:360-365. 4. Cardella, M. A., J. T. Duff, B. H. Wingfield, and C. Gottfried. 1959. Studies on immunity to toxins of Clostridium botulinum. VI. Purification and detoxification of type D and immunological response to toxoid. J. Bacteriol. 79:372-378. 5. Gordon, M., M. A. Fiock, A. Yarinsky, and J. T. Duff. 1957. Studies on immunity to toxins of Clostridium
6.
7.
8.
9. 10.
botulinum. III. Preparation, purification, and detoxification of type E toxin. J. Bacteriol. 74:533-538. Kerr, S. E., and K. Seraidarian. 1945. The separation of purine nucleoside from free purines and the determination of the purines and ribose in these fractions. J. Biol. Chem. 159:211-225. Kitamura, M., S. Sakaguchi, and G. Sakaguchi. 1968. Purification and some properties of Clostridium botulinum type-E toxin. Biochim. Biophys. Acta 168: 207-217. Kozaki, S., S. Sakaguchi, and G. Sakaguchi. 1974. The purification and some properties of progenitor toxins of Clostridium botulinum type B. Infect. Immun. 10:750-756. Lamanna, C., 0. E. McElroy, and H. W. Eklund. 1946. The purification and crystallization of Clostridium botulinum type A toxin. Science 103:613-614. Lowry, 0. H., N. J. Rosebrough, A. L Farr, and R.
VOL. 19, 1978 J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 11. Miyazaki, S., ML Iwasaki, and G. Sakaguchi. 1977. Clostridiun botulinum type D toxin: purification, molecular structure, and some immunological properties. Infect. Immun. 17:395-401. 12. Ohishi, I., and G. Sakraguchi. 1974. Purification of Cbostridium botAdinum type F progenitor toxin. Appl. Microbiol. 28:923-928.
NOTES
751
13. Snipe, P. T., and H. Sommer. 1928. Studies on botulinus toxin. 3. Acid precipitation of botulinus toxin. J. Infect. Dis. 43:152-160. 14. Sugii, S., and G. Sakaguchi. 1974. Molecular construction of Closhtidiwn botulinum type A toxins. Infect. Immun. 12:1262-1270. 15. Vinet, G., and IL Raynaud. 1963. Preliminary note on production and purification of botulinal toxin type C. Rev. Can. Biol. 22:119-120.
Vol. 19, No. 2
0019-9567/78/0019-0749$02.00/0 Copyright X 1978 American Society for Microbiology
Printed in U.S.A.
Acid Precipitation of Clostridium botulinum Type C and D Toxins from Whole Culture by Addition of Ribonucleic Acid as a Precipitation Aid MASAKAZU IWASAKI AND GENJI SAKAGUCHI* Department of Veterinary Science, College of Agriculture, University of Osaka Prefecture, Sakai-shi, Osaka
591, Japan Received for publication 15 September 1977
The ratios of ribonucleic acid to protein contents of Clostridium botulinum type C, D, and E cultures were lower than those of type A, B, and F cultures. Addition of ribonucleic acid at 0.4 mg/ml to culture satisfactorily aided acid precipitation of type C and D toxins, but not that of type E toxin. C. botulinum type A toxin has been obtained in crystalline form (1, 9). Type A, B, and F toxins have been purified by similar procedures in this laboratory (8, 12, 14). In purification of these toxins, acid precipitation was used as the first step to concentrate the toxin from whole culture. This procedure is relatively simple, safe, and inexpensive to perform when handling a large volume of culture. Type C, D, and E toxins have also been purified by investigators in this and other laboratories (3-5, 7, 15), but acid precipitation was not applied because of the poor recovery of toxin (3, 5). It is known that type A, B, and F toxins themselves are soluble at pH 4.0, but are precipitable at this pH only when they are in crude form. After treating the acid precipitate with protamine or ribonuclease, the toxin became soluble at pH 4.0 (8, 12, 14). Nucleic acids or some other acidic substances must have played an important role in the acid precipitation of the toxin (13). We attempted, therefore, to determine the ribonucleic acid (RNA) contents of cultures of types A through F and to add RNA to type C, D, and E cultures that contained less RNA than type A, B, and F cultures to see if it would aid acid precipitation of the toxins. Each strain of C. botulinum type A through F, the names of which are given in Table 1, was grown in a medium consisting of 2% peptone, 0.5% yeast extract, 0.5% glucose, and 0.1% Lcysteine-hydrochloride, pH 7.2, for 3 days at
300C. Protein contents were determined according to Lowry et al. (10) on the residue of cold and then hot 5% trichloroacetic acid extraction. RNA contents were determined according to Kerr and Seraidarian (6) on the cold trichloroacetic acid precipitate. Protein and RNA contents of cultures of type A through F are shown in Table 1. The ratio of
RNA to protein contents of type C, D, and E cultures were apparently lower than those of type A, B, and F cultures. We presumed that the toxin would be precipitated by acidification if the RNA-to-protein ratio of culture was made to 0.77 or higher. RNA (Yeast RNA, P-L Biochemical Inc., Milwaukee, Wis.) was dissolved in 2 N NaOH. The RNA solution was adjusted to pH 6.0 with 1 N HCI and brought to a final concentration of 10 mg/ml by the addition of distilled water. The cultures of types C, D, and E were each divided into two portions. To a portion of each culture, the RNA solution was added to a concentration of 0.4 mg/ml. Both portions were adjusted to pH 4.0 with 3 N H2SO4. The acidified cultures were allowed to stand for 3 h at room temperature. The precipitate formed was collected by centrifugation for 5 min at 1,500 x g and 4°C and resuspended in 0.2 M phosphate buffer, pH 6.0, to the original volume. Toxin was titrated by the time-to-death method by intravenous injection of a fourfold dilution into mice (2). Type B, E, and D (strain CB16 only) toxins were activated with trypsin before injection (7, 8, 11). The toxicities of acid precipitate obtained in the presence and absence of RNA are shown in Table 2. Without the aid of RNA, the recovery of the toxin was not satisfactory. In the presence of RNA at 0.4 mg/ml, the recovery of the toxin was satisfactorily high with all type C and D strains. Little or no toxin remained in the supernatant. The recovery of toxin was not satisfactory when RNA was added to 0.2 mg/ml or lower concentrations. With type E culture, acidification in the absence of RNA recovered 62% of the toxin in the centrifugal precipitate. This was due apparently to the association of type E toxin with the bacterial cells (7). Addition of RNA did not increase 749
INFECT. IMMUN.
NOTES
750
Type C C
TABLE 1. Protein and RNA contents of whole cultures of types A through F RNA (pg/ml) RNA/protein ratio Strain Protein (pg/ml) CB19 203
630 681
126 115
0.20 0.17
Stockholm
353
35
0.10
1873 CB16 4947
630 382 658
85 40 245
0.13 0.10 0.37
E
German sprats
438
244
0.55
A B B F
62
345
377
1.09
Okra
337
460
1.36
QC
388
346
0.89
Langeland
332
254
0.77
C D
D D
TABLE 2. Acid precipitation of type C, D, and E toxins with or without added RNA
LDgia/ml X 10-4 in: b Acid precipitate
Strain
Type
Whole culture RNA not added (%)
a
C C
CB19 203
C D D
Stockholm 1873 CB16
D
4947
E B
German sprats Okra
RNA added (%)
(35) (50)
19 35 170
22
(45) (17) 6.9 (31)
16
7.3 (46)
15
26 36 0.77 200
9.1 18 0.35 34
5.6 (62) 220 (92)
9.0 240
(73) (97)
0.64 (83) (85) 18 (82) (94)
6.0 (67) NDC
LD5o, 50% lethal dose.
b Numbers indicate the absolute amounts of toxin recovered. c
ND, Not done.
the recovery of the toxin in the precipitate. Type E toxin is adsorbed onto CM-Sephadex at pH 6.0 (7), whereas type B toxin is not under the same conditions (8). Type E toxin released from the bacterial cells, therefore, may differ in electrostatic property from toxins of other types. The extract of the acid precipitate of type C and D toxins obtained by the aid of RNA contained a large quantity of RNA, but the RNA was removed subsequently by the protamine treatment (8, 12, 14) without appreciable loss of the toxin. We have succeeded in purifying type C and D (11) toxins by applying the same subsequent procedures, namely chromatography on SP-Sephadex C-50 and molecular sieving on Sephadex G-200, as used for purifying type A, B, and F toxins (8, 12, 14). Thus, acid precipitation of type C and D toxins from whole culture in the presence of RNA added as a precipitation aid is very effective as the initial step of purification from large quantities of type C and D cultures. LITERATURE CITED 1. Abrams, A., G. Kegeles, and G. A. Hottle. 1946. The purification of toxin from Clostridium botulinum type A. J. Biol. Chem. 164:63-79.
2. Boroff, D. A., and U. Fleck. 1966. A statistical analysis of rapid in vivo method for the titration of the toxin of Clostridium botulinum. J. Bacteriol. 92:1580-1581. 3. Cardelia, M. A., J. T. Duff, C. Gottfried, and J. S. Begel. 1958. Studies on immunity to toxins of Clostridium botulinum. IV. Production and purification of type C toxin for conversion to toxoid. J. Bacteriol.
75:360-365. 4. Cardella, M. A., J. T. Duff, B. H. Wingfield, and C. Gottfried. 1959. Studies on immunity to toxins of Clostridium botulinum. VI. Purification and detoxification of type D and immunological response to toxoid. J. Bacteriol. 79:372-378. 5. Gordon, M., M. A. Fiock, A. Yarinsky, and J. T. Duff. 1957. Studies on immunity to toxins of Clostridium
6.
7.
8.
9. 10.
botulinum. III. Preparation, purification, and detoxification of type E toxin. J. Bacteriol. 74:533-538. Kerr, S. E., and K. Seraidarian. 1945. The separation of purine nucleoside from free purines and the determination of the purines and ribose in these fractions. J. Biol. Chem. 159:211-225. Kitamura, M., S. Sakaguchi, and G. Sakaguchi. 1968. Purification and some properties of Clostridium botulinum type-E toxin. Biochim. Biophys. Acta 168: 207-217. Kozaki, S., S. Sakaguchi, and G. Sakaguchi. 1974. The purification and some properties of progenitor toxins of Clostridium botulinum type B. Infect. Immun. 10:750-756. Lamanna, C., 0. E. McElroy, and H. W. Eklund. 1946. The purification and crystallization of Clostridium botulinum type A toxin. Science 103:613-614. Lowry, 0. H., N. J. Rosebrough, A. L Farr, and R.
VOL. 19, 1978 J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 11. Miyazaki, S., ML Iwasaki, and G. Sakaguchi. 1977. Clostridiun botulinum type D toxin: purification, molecular structure, and some immunological properties. Infect. Immun. 17:395-401. 12. Ohishi, I., and G. Sakraguchi. 1974. Purification of Cbostridium botAdinum type F progenitor toxin. Appl. Microbiol. 28:923-928.
NOTES
751
13. Snipe, P. T., and H. Sommer. 1928. Studies on botulinus toxin. 3. Acid precipitation of botulinus toxin. J. Infect. Dis. 43:152-160. 14. Sugii, S., and G. Sakaguchi. 1974. Molecular construction of Closhtidiwn botulinum type A toxins. Infect. Immun. 12:1262-1270. 15. Vinet, G., and IL Raynaud. 1963. Preliminary note on production and purification of botulinal toxin type C. Rev. Can. Biol. 22:119-120.
