Chemical Studies on Tuberculin

voL. 11, 1925

BACTERIOLOGY: J. H. MUELLER

23

CHEMICAL STUDIES ON TUBERCULIN By J. HowARD MUZLLIR DsPARTMJNT OF BACTZRIOLOGY, HARVARD MIDICAL SCROOL Read before the Academy November 12, 1924

In his earlier work with tuberculosis, Zinsser showed that an alkaline extract of tubercle bacilli, rendered as free as possible from protein material was capable of producing a typical skin reaction of the tuberculin type in tuberculous guinea pigs. A similar skin reaction was also produced by the acid-insoluble protein fraction separated from the extract during the process of purification. At that time it could not be determined whether this was due to the adsorption of some of the so-called residue or protein-free fraction by the protein precipitate, the presence in the residue of small quantities of unprecipitated protein, or to the splitting off of an active non-protein fraction from a similarly active protein mother substance. However, the fact that an active tuberculin, precipitable by alcohol, but free from, or at least extremely low in, protein, could be prepared, was sufficiently striking to warrant extending the observation to other bacteria to determine whether or not the phenomenon was general. During the course of subsequent experiments, Zinsser and Parker showed that extracts could be prepared by the same method from all, types of bacteria investigated, including the pneumococcus, meningococcus, influenza bacillus and staphylococcus. These extracts were tested, however, by the precipitin test with specific immune sera, instead of the skin reaction, because of the difficulty of producing chronic infections in guinea pigs with these organisms. In each case the residue material precipitated specifically. The writer undertook an investigation of these extracts from the chemical standpoint to determine if possible whether they contained protein and owed their specific serological reactions to that fact, or whether some hitherto -unsuspected type of compound was capable of eliciting immune reactions. In collaboration with Zinsser and Waytnan it was at first shown that the active material precipitated by alcohol fiom the residue extracts, and redissolved in such concentration that mere dilution could not account for negative results, gave none of the usual color tests for protein, except the xanthoproteic reaction which occasionally appeared positive, but so slightly as to be of doubtful significance. Nitrogen was next determined on the precipitated and dried material and was found present to the extent of from one to three per cent. The quantities of residue obtainable by the facilities at our disposal were so small as to make it impossible to determine in what form this existed. The non-nitrogenous material appeared to be, in part at least, carbohydrate since it gave a positive Molisch reaction.

24

BACTERIOLOGY: J. H. MUELLER

PROC. N. A. S.

In casting about for a method of attacking the problem in a manner which might hold some promise of success, we attempted to extend the investigation to a study of a similarly prepared extract of bread yeast, since this was readily obtainable in large quantities. This proved to be possible and with Tomscik we were able to prepare by a slightly modified method, considerable quantities of a complex carbohydrate, or gum, corresponding in the general method of preparation and in properties with the yeast gum or Hefegummi of the literature. This gives a specific precipitation reaction in high dilution with anti-yeast serum. It contained in some preparations less than one per cent of nitrogen and because of various considerations we felt reasonably sure that it was the carbohydrate itself which was specifically active. During the course of these experiments, Heidleberger and Avery's work appeared, in which a similar complex carbohydrate was isolated by them from Type II Pneumococcus cultures, which also gave a specific precipitin test in extremely high dilutions. With the experience gained in the yeast work, and with the additional aid of Heidleberger and Avery's methods, an investigation of the nature of the active material of the tuberculin is now under way. Specific precipitating sera prepared by injecting rabbits with dead tubercle bacilli are now available, and while in the case of the earlier work on tubercle extracts, to which reference has been made, the skin test alone was used as an indication of activity, 'both tests are now being run in parallel. While it was assumed that the same component of the tuberculin was responsible for both types of reaction, this has apparently proved not to be the case. It has been possible to obtain a fraction of tuberculin containing about one per cent nitrogen, giving a specific .ring precipitin test in dilutions up to 1-400,000 with immune serum, and yet without any ability to produce a skin reaction in concentrations corresponding roughly to highly potent quantities of the original tuberculin. This fraction contains carbohydrate and is apparently comparable to the yeast gum, and to Heidelberger and Avery's pneumococcus preparations. On the other hand, a second fraction has been separated, giving a specific precipitin reaction in low dilutions only, but producing a specific skin test in tuberculous animals in quantities equivalent to active concentrations of tuberculin. This fraction gives a strong biuret reaction in concentrated solutions, but it is not yet possible to say whether the material is even moderately pure or not. One may perhaps suspect that the substance producing the tuberculin action will prove to be either protein or some protein split product, for while the evidence in the literature is conflicting, it seems to have been pretty well shown, and our own experiments have confirmed the fact, that proteolytic enzymes destroy the active material rather quickly. On the other

VOiL. 1 1, 1925

PHYSICS: ALLISON AND DUANE

25

hand we have been able to show that the specific precipitable material of the tuberculin, like that of the yeast, is resistant to the action of proteolytic enzymes, and Heidleberger and Avery have demonstrated the same fact for their pneumococcus preparations. Work on both these fractions is being continued in our laboratory to the end of clearing up their chemical nature and arriving at an understanding of the part each plays in the disease processes.

ON SCATTERED RADIATION DUE TO X-RA YS FROM MOL YBDENUM AND TUNGSTEN TARGETS By S. K. ALLISON' AND WILLIAM DUAN1

JsrrBPRSON PHYSICAL LABORATORY, HARVARD UNIVERSITY Communicated December 5, 1924

In notes recently published in these PROCrDINNfs experiments performed in our laboratory have been described in which the spectra of secondary x-radiation have been examined by an ionization x-ray spectrometer. The curves representing the ionization currents as functions of the glancing angles of incidence of the rays on the calcite reflecting crystal contained sharply marked peaks representing scattered radiation from the secondary radiator having wave-lengths equal to those of the primary rays to within the limits of error of the measurements, but no peaks appeared indicating radiation comparable in intensity with the above scattered radiation and having wave-lengths corresponding with Professor A. H. Compton's interesting and important theory of single electron scattering. Recently we have repeated some of these experiments with two important modifications in the apparatus and have found evidence of radiation agreeing very well with the equation

AX = 0.024 (1 - cos 0),

(1)

which Professor Compton deduced from his theory. Figure 1 on page 381 of the September number of the Proceedings of the National Academy of Sciences represents the general arrangement of the apparatus. No large masses lay near the secondary radiator and x-ray tube except the unavoidable lead plate fastened against the brick wall. The most important changes in the spectrometer are as follows: (a) Instead of employing a water-cooled molybdenum target tube with a large bulb blown in it, we used in some experiments a water-cooled molybdenum target sealed into a glass tube of approximately uniform diameter throughout. This tube was about 3 cm. in diameter and its walls were made