The Journal of Biochemistry, Vol. 28, No. 3. ON THE NUCLEIC ACID OF TYPHOID BACILLUS OF MICE. BY SYUZO AKASI. (From the Medico-chemical Institute, Kyoto Imperial University. Director: Prof. K. Mayeda.) (Received for publication, June 20, 1938) We have little information regarding the nucleic acid of bacteria, whether concerning a pathogenic or a non-pathogenic bacteria. Studies of nucleic acids have been made with coli bacillus by Schaffer, Forkoff, and Jone (1922), with azotobacter by Mockeridge (1924), with tubercle bacillus by Ruppel (1898-99) and later by Johnson and Brown (1922 & 1923), and with timothy bacillus (1931) as well as diphtheria bacillus (1932) re cently by Coghil1. All the workers were truly in accord with the finding that bacterial cells contain generally a nucleic acid as a cell constituent, but the individual results indicated a marked difference from each other, so that as to the question whether it deals with the animal or the plant type of nucleic acid, or in another term, lately introduced, a ribose or a desoxyribose nucleic acid, a definite answer has not yet been offered. For the purpose of aquiring a clear knowledge of this problem many detailed in vestigations extending also to various bacilli are desired , notwith standing the fact that in this field of study many difficulties are naturally encountered, the foremost being to prepare a sufficient amount of investigation materials from bacteria, in whose cultiva tion enormous expense is involved, and secondly, to submit the crude product to a rigorous purification for detailed chemical analysis. The present paper reports the study of nucleic acid of typhoid bacillus of a mouse, which was conducted with the aim of obtaining material from bacilli in a high state of purity and to make a precise analysis of it in many respects, so that a com 355 356 S. Akasi: parison of results with those of the other known nucleic acids might be drawn. EXPERIMENTAL. Cultivation and Preparation of Bacillus Material. The typhoid bacillus of a mouse of the strain, Tohoku 101, with which the writer was supplied for study, was cultivated and collected in the following way: It was grown by means of a special cultivation vessel equipped with the solid culture medium com posed of 25.0gm. of condensed extract of bonito fish, 25.0gm. of Teruuchi's peptone, 30.0gm. of agar-agar, and 1000cc . of water. After keeping the inoculated cultivation vessel for 48 hours in an incubator at 38•‹, the grown bacilli were washed out of the vessels with distilled water containing 0.5% by volume of formaline, and all the washing fluids holding bacilli in suspension were brought together into the stoppered bottles , which were then kept for 72 hours in an incubator at 37•‹ so as to secure a perfect sterilization . The combined fluids were then passed through a Sharple's centri fuge, which effected the separation of bacilli from the washing fl uid. The bacilli thus obtained in the form of a thick paste were spread upon the sterilized absorption plates , dried in vacuo at 45•‹ and finally ground to a slightly yellowish , white powder. This bacillus material, weighing 500gm ., was at first extracted exhaustively with ether in order to remove the fatty substance , the analytical research of which will be given in a future publica tion, and then the fat-freed bacilli were employed for the prepara tion of nucleic acid as follows. Isolation of Crude Nucleic Acid . This was carried out following essentially the method which Coghi11 used for the preparation of nucleic acid from timothy grass hay bacillus. 450gm. of bacteria, as given above, were ground in a mortar with 2 litres of 2% sodium hydroxide solution to make a thick paste and allowed to stand an hour at room temperature. Then 2 On the Nucleic Acid of Typhoid Bacillus of Mice. 357 litres of water were added, mixed thoroughly, and treated with acetic acid until a neutrality towards lithmus was reached. The mixture was transferred in a centrifuge bottle and centrifuged. The strong turbid supernatant fluid was separated from the re sultant precipitate, which was washed once by suspending in 2.5 litres of distilled water and again centrifuged. The combined filtrate and washings were treated with dilute colloidal ferric hydroxide solution with vigorous stirring in order to get a smooth filtration by filtering the mixture off through fluted filter papers. The clear filtrate was acidified with hydrochloric acid to an acid reaction to congo red indicator. Then an equal volume of alcohol was added and the precipitated nucleic acid was centrifuged and treated with alcohol and ether. The crude nucleic acid, thus ob tained, weighed 20gm. when dried in the air. This substance was not, however, in so high a state of purity as described by Coghill in the case of timothy bacillus, and judging from the strong biuret reaction observed, it seemed to contain a considerable amount of proteins. The preliminary test of purification with a small sample was carried out in an ordinary manner, namely, by dissolution in alkaline and reprecipitation with acid. This attempt was, how ever, unsuccessful, since even after five repetitions of a similar procedure the sample could not be rendered entirely free from biuret test, and in addition each purification procedure involved a serious loss of materials. On this account an easy purification method which leads to the production of pure nucleic acid was developed, as described in the following. Purification of Crude Nucleic Acid. 15gm. of crude nucleic acid were dissolved in 200cc. of 10%, sodium acetate solution and 5 volumes of glacial acetic acid were added. A small quantity of insoluble matter was removed by cen trifugation and the filtrate was treated with enough 10% copper chloride solution to cause nucleic acid as a copper salt to flock out. After standing a short time, the precipitate was centrifuged and washed twice with glacial acetic acid. Then the copper salt was decomposed by treating with acetone containing hydrochloric acid 358 S. Akasi: in 5% and the acetone solution was discarded after centrifugation. This treatment was twice repeated in a similar manner. The precipitate of almost white color finally obtained was suspended in a little water and dissolved by the addition of a small amount of sodium hydroxide solution. After eliminating the slight in soluble matter by centrifugation the clear solution was acidified with hydrochloric acid until acid reaction towards congo red was attained. This solution was transferred into centrifuge bottles and upon addition of two volumes of alcohol, the separated precipitate of the free nucleic acid was centrifuged. It was then treated with alcohol and ether and finally dried in the air. The yield was 5.5gm., corresponding therefore to 1.6 Jo of the original bacilli. It formed a white, non-hygroscopic powder, and was easily soluble in concentrated acetic acid. The concentrated aqueous solution of its sodium salt presented neither a gelatinous nor a viscous fluid, as is usually the case with sodium salt of thymonucleic acid. The biuret reaction was not proved in the ordinary.manner as well as by the special technique of Osborne-this was done by operating the test in the usual way, then adding 10 or 20 drops of ethyl alcohol and a stick of solid potassium hydroxide, when the alkali salts out the alcohol, which brings with it any of the biuret color, if present. With a pure sample both the phloroglucinol and orcinol tests for pentose were proved and further a positive Shiff's fuchsin sulfurous acid reaction or Feu1gen's nuclear reaction was also obtained. Analysis of the Pure Nucleic Acid. For analysis the air-dry material was further dried in vacuo at 100•‹ to a constant weight, when it was tinged somewhat with a light brown color. Nitrogen Content-(Kjeldahl's method). 3.452mg. substance: 3.70cc. 0.01n HCl; 14.63% N. 5.254 ,, ,,: 5.40 ,, ,, ,,; 14.39,, ,, . Found. average 14.51 ,, ,,. Phosphorus Content-(Pregl-Lieb's method). 11.321mg. substance: 71.022mg. ammonium phosphomolybdate; 9.07% P. On the Nucleic Acid of Typhoid Bacillus of Mice. 359 5.980 ,, ,,: 37.733 ,, ,, ,,; 9.01 ,, ,, Found. average 9.04 ,, ,, (Conversion factor=0.014525) Hence Total-N: Total-P=1.61:1 Pentose Content-The determination of pentose was executed employing the method described by Hoffman (1927). The sample was transferred into a 500cc. distilling flask made according to the directions of the author. 50 cc. of 20% hydrochloric acid were added and distilled with a current of steam for three hours, always keeping the temperature of the reaction mixture constant at 103 - 105•‹. The distillate, collected in a measuring flask, was titrated in this flask with 10% sodium hydroxide from a burette to neutrality to phenolphthalein, and the liquid was then diluted to an adequate volume, in the present case 700cc. The standard solution containing furfural approximately equal to that expected in the distillate was placed in a flask of the same size and treated with hydrochloric acid and sodium hydroxide until the solution was neutral to phenolphthalein, care being taken that the sodium hydroxide added might equal the amount added to the unknown. This solution was also diluted to the mark. 6cc. of each test and standard solution were transferred to a test tube, each was treated with 0.5cc. of aniline and 4.0cc. of glacial acetic acid, allowed to stand for 10 to 15 minutes in the dark, and then compared in a colorimeter. The amount of pentose was caluculated by multi plying the obtained value by a conversion factor=1.98 in ac cordance with Coghill (1931), the latter being the average value given for xylose and arabinose by Youngburg (1927).