A method for preparation of antibacterial basic proteins of normal tissues.

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.