A METHOD FOR PREPARATION OF ANTIBACTERIAL BASIC ...

A METHOD FOR PREPARATION OF ANTIBACTERIAL BASIC PROTEINS OF NORMAL TISSUES WALTER LYON BLOOM AND JOHN R. PRIGMORE Departments of Biochemistry and Medicine, Emory University School of Medicine, Emory University, Georgia Received for publication May 8, 1952

MATERIALS AND METHODS

Fresh beef spleen (5 pounds) was trimmed and ground with a coarse meat grinder. The ground spleen was treated with an equal volume of 0.25 N H2SO4 855

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The possibility that normal tissue constituents may be involved in the mechanism of resistance to infection with Bacillus anthracis was first considered in 1890. In that year Hankin reported the isolation of germicidal extracts from the spleen and lymphatic glands of cats. In 1893 Vaughn reported the preparation of germicidal extracts from spleen, brain, thyroid, and testicular tissue. His procedure, however, varied markedly from that of Hankin, and a "nuclein" fraction was thought to contain the germicidal substance. Active experimentation with tissue extracts in the study of natural immunity toward anthrax was infrequent after this earlier interest. This is surprising since it was already known that the naturally resistant host presents an unfavorable environment for the survival of the invading parasite. In most instances natural resistance must depend upon the presence of chemical entities in sufficient concentration to create an unfavorable environment for continued parasite proliferation. In 1947 (Bloom, Watson, Cromartie, and Freed) further findings in this phase of microbiology were elaborated. During the course of investigations on anthrax these authors succeeded in extracting active anthracidal fractions from the cecum, thymus, and pancreas of various animal species. Although some purification of the active material was effected by ethanol fractionation, even the crude extract was seen to exert almost immediate anthracidal activity. Chemical and electrophoretic studies indicated that the substance was a basic polypeptide containing a high content of lysine. Bloom and Blake (1948) found an active fraction in still another tissue, thyroid. In this study these antibacterial fractions were shown to have in vitro activity when tested with Escherichia coli, Staphylococcus aureus, beta hemolytic streptococci, and Bacillus megaterium. Recently Bloom, Winters, and Watson (1951) reported on the antibacterial effects of the thymus basic protein and protamine on Bacillus subtilis in vitro. In devising a suitable method for these extractions, a somewhat lengthy procedure was previously elaborated. The present paper suggests a simplification of the original method of preparation of the antibacterial substances and, for the first time, reports the isolation of an active fraction from the spleen. A comparative quantitative study of the antibacterial action of fractions from the spleen, thymus, and pancreas on Bacillus subtilis is presented.

856

WALTER L. BLOOM AND

JOHN

R.

PRIGMORE

[VOL. 64

RESULTS

In order to test the activity of these preparations, Warburg studies were carried out on aliquots of the filtrates and the precipitates removed at various phases of the spleen preparation. The findings are presented in table 1 and show that no measurable activity was lost in rejected materials.


and allowed to stand for 4 days at room temperature for extraction of the polypeptide. A slow increase in pH necessitated daily readjustment to the desired pH of 1.5. After extraction the tissue was filtered, first through cheese cloth and then through cotton. Then the pH of the solution was adjusted to 3.5 with 5 N NaOH. Aliquots of 250 ml were removed to one liter Erlenmeyer flasks and heated, with constant stirring, in a boiling water bath. After an internal flask temperature of 90 C had been reached, the heating was continued for exactly 5 minutes. The solutions were permitted to cool to a temperature of 50 C, and the flocculent precipitate was filtered off in the cold room at 3 to 5 C through a fluted Whatman no. 12 filter paper of 38.5 cm diameter. The precipitate was discarded. The cold filtrates were combined and mixed with an equal volume of cold acetone and allowed to stand overnight at a temperature of 3 C. The major portion of the supernatant liquid was siphoned off then, and the remainder of the material was filtered through a fluted 38.5 cm Whatman no. 12 filter paper. A portion of the rejected supernatant was retained for later examination and designated as "acetone supernatant, S-I". The white precipitate was suspended in one-tenth of the original volume of 0.85 per cent NaCl and dialyzed for 60 hours in the cold at 3 C against distilled water (changed every 12 hours). The dialyzate was adjusted to pH 7.0 with N NaOH. The solution was filtered, then centrifuged, and the precipitate discarded. A 10 ml aliquot of this filtrate was removed for later examination, frozen, lyophilized, and designated as "acetone precipitate, S-II". The remainder of the filtrate was dialyzed for 48 hours in the cold at 3 C against 0.1 M acetate buffer, pH 3.5 (changed every 12 hours), and then centrifuged. The supernatant solution at pH 3.7 was cooled to -2 C, and 95 per cent ethyl alcohol was added dropwise, with constant stirring, to a final concentration of 40 per cent alcohol by volume. The white precipitate which formed was separated by centrifugation, washed with cold water, dissolved in 0.85 per cent NaCl, dialyzed for 48 hours in the cold at 3 C against distilled water, and set aside for later examination with the designation "alcohol precipitate, S-III". The supernatant was dialyzed for 72 hours in the cold against distilled water and then was frozen and lyophilized, yielding a white, water soluble product, very light in weight. The final product was designated as "40 per cent alcohol supernatant, S-IV" The strain of B. subtilis was isolated in this laboratory and was maintained by frequent transfer in nutrient broth. The Warburg studies were conducted as previously described (Bloom, Watson, Cromartie, and Freed, 1947). Reproducibility of results was confirmed by repeating each Warburg experiment on three different occasions.

.1952]

PREPARATION OF ANTIBACTERIAL BASIC PROTEINS8857

In an effort to determine the highest dilution of the spleen fraction which would cause complete cessation of oxygen uptake, an assay by serial dilution was carried out. The results are shown in table 2. One hundred and seventy-five micrograms represent the minimal amount of bactericidal substance in the spleen which completely inhibits oxygen uptake under the experimental conditions employed. This represents a concentration of 58 ,g per ml of culture in the Warburg flask.

B. SUBTILIS 02 UPTAKE IN CM PER 10 mIIN TlrVALS CONTNTS

A. B. C. D. E.

OF SID

ARM

Acetone supernatant, S-I Acetone precipitate, S-II 40% alcohol precipitate, S-III 40% alcohol supernatant, s-IV Control (saline added)

I

II

III

IV

V

VI

VII

0.2 0.05 0.15 0.05 0.3

0.8 0.75 1.0 0.75 0.8

1.8 1.8 1.9 1.8 1.4

3.2* 3.3* 3.3* 3.3* 3.5*

5.2 3.4 5.8 3.3 5.0

8.1 3.4 8.9 3.3 8.6

11.7 3.4 12.0 3.3 13.1

* Periods I-IV, control observations. Material under test was tipped in from the side arm after period IV, and the reaction was studied for 3 subsequent periods.

TABLE 2 Effect of various amounts of lyophilized spleen preparation, alcohol supernatant, S-IV, on the oxygen uptake of Bacillus subtilis AMUtONT OP BASIC POLYPPTDE I SIDE R (MICROGRAMS)

A. B. C. D. E. F.

250 200 175 150 100 Control (saline added)

B.

SUBTILS 02 UPTA31 IN CM PEL 10 mN INTERVALS

I

0.35 0.25 0.15 0.05 0.2 0.6

0.7 0.8 0.8 0.9 1.0 1.2

III

IV

V

VI

1.75 2.3 1.6 1.9 2.05 2.0

3.15* 4.1* 3.2* 3.9* 4.05* 3.7*

3.15 4.1 3.2 4.65 6.0 5.1

3.15 4.1 3.2 7.4 8.7 8.2

VII

3.15 4.1 3.2 11.3 13.1 12.7

* Active material added from side arm after this period.

The above procedure was also employed for the isolation of active antibacterial fractions from beef pancreas and calf thymus. Under our experimental conditions, 200 ,g represent the mi imal concentration of biological activity in the Warburg apparatus for the pancreas preparation (or 67 ,g per ml of culture in the flask). The thymus fraction proved active to 250 Mug (or 83 Mug per ml of culture in the flask). DISCUSSION

The development of a simplified method for the preparation of au antibacterial protein of normal tissues should expedite the study of this type of protein. The


TABLE 1 The effect of addition of representative fractions of spleen preparation on oxygen uptake of Bacillus subtilis

858

WALTER L. BLOOM AND JOHN R. PRIGMORE

[VOL. 64

SUMMARY

The occurrence of antibacterial substances in beef pancreas and thymus tissue has been further studied, and the preparation of such material in spleen has been demonstrated for the first time. A simplified method of preparation of these substances has been developed. REFERENCES BLOOM, W. L., AND BLAKE, F. G. 1948 Studies on an antibacterial polypeptide extracted from normal tissues. J. Infectious Diseases, 88, 116-123. BLOOM, W. L., WATSON, D. W., CROMAiTIE, W. J., AND FREED, M. 1947 Studies on infection with Bacillus anthracis. IV. Preparation and characterization of an anthracidal substance from various animal tissues. J. Infectious Diseases, 80, 41-52. BLOOM, W. L., WINTERS, M. G., AND WATSON, D. W. 1951 The inhibition of two antibacterial basic proteins by nucleic acids. J. Bact., 62, 7-13. HANKIN, E. H. 1890 A bacteria-killing globulin. Proc. Roy. Soc. (London), 48, 93-101. KOSsEL, ALBRECHT 1928 The protamines and histones. Longmans, Green and Company, New York, N. Y. STAHMANN, M. A., GRAF, L. H., PATTERSON, E. L., WALKER, J. C., AND WATSON, D. W. 1951 The inhibition of tobacco mosaic virus by synthetic lysine polypeptides. J. Biol. Chem., 189, 45-51. VAUGHN, V. C., AND MCCLINTOCK, C. T. 1893 The nature of the germicidal constituents of blood-serum. Med. News Phila., 68, 701-707. WATSON, D. W., AND BLoom, W. L. 1952 Unpublished data. WEISSMAN, W., AND GRAF, L. H. 1947 Studies on infection with Bacillus anthracis. VII. A comparison of the antibacterial effects of calf thymus histone and a quarternary ammonium cationic detergent on B. anthraci8. J. Infectious Diseases, 80, 145-153.


material has many of the characteristics of the proteins classed as histones (Kossel, 1928), but previous study (Weissman and Graf, 1947) has demonstrated differences in the quantities of histone (prepared by classical procedures) and of antibacterial proteins necesary to inhibit bacterial oxygen uptake. In the present investigation, quantitative differences have been demonstrated in the antibacterial properties of the preparations from thymus, pancreas, and spleen of the bovine species. The probable nuclear origin of this material (Bloom, Watson, Cromartie, and Freed, 1947) suggests that any cellular tissue may be an adequate source of material. It has not been demonstrated that the differences in activity are related to the concentration of basic amino acids in the molecule of basic protein. However, the recent studies of Stahmann et at. (1951); Bloom, Winters, and Watson (1951); and Watson and Bloom (1952) indicate that the activity of these preparations may be related to the basic amino acid content of the protein.