ADENINE-URACIL DINUCLEOTIDE AND THE STRUC

TURE OF YEAST NUCLEIC ACID.

BY WALTER JONES AND B. E. READ.

(From the Laboratory of Physiological Chemistry, Johns Hopkins University, Baltimore.)

(Received for publication, December 27, 1916.)

Recent investigations have necessitated the assumption that yeast nucleic acid contains groups of the four following mononucleotides.1-7

O PO. CH ̧О ̧ . С¿H¿Ñ¿O guanine mononucleotide.
HO

The gross structure of nucleic acid would therefore be determined if we could discover the points at which the four nucleotide groups are joined to one another. Only two modes of linkage have sufficient probability to require discussion. One is through the phosphoric acid groups, and the other is through the carbohydrate groups. In the one case, nucleic acid would be a substituted polyphosphoric acid, and in the other case, a substituted

1 Levene, P. A., and Jacobs, W. A., Ber. chem. Ges., 1909, xlii, 2474.

2 Levene and Jacobs, Ber. chem. Ges., 1909, xlii, 2703.

Levene and Jacobs, Ber. chem. Ges., 1910, xliii, 3150.

5

Levene and Jacobs, Ber. chem. Ges., 1911, xliv, 1027.

Levene, P. A., and La Forge, F. B., Ber. chem. Ges., 1912, xlv, 608.

Jones, W., and Richards, A. E., J. Biol. Chem., 1914, xvii, 71.

7 Jones and Richards, J. Biol. Chem., 1915, xx, 25.

polysaccharide. Prevailing opinion favors the former view, and nucleic acid has been assigned the formula:

The following considerations, however, prove that this is not the correct solution. We have prepared from yeast nucleic acid a substance which by acid hydrolysis yields adenine and uracil but neither guanine nor cytosine. By ammoniacal hydrolysis it yields adenosine and uridine but neither guanosine nor cytidine. The partition of its phosphorus shows that it contains an equal number of purine and pyrimidine groups (one of each). The substance is evidently adenine-uracil dinucleotide. Its physical properties are strikingly different from those of yeast nucleic acid and the yield is such as to suggest that its formation from yeast nucleic acid is quantitative.

When a hot aqueous solution of the dinucleotide is treated with a solution of brucine in hot alcohol, a tetrabrucine salt is formed which is deposited in beautifully crystalline form as the solution cools. Neither the melting point nor the elementary composition of the brucine salt is changed by repeated crystallization of the substance from hot water. By acid hydrolysis it yields adenine but not guanine. Half of its phosphoric acid is easily split, half is firmly bound. Analyses for carbon, hydrogen, nitrogen, phosphorus, and brucine give sharply the values required for the formula C19H25N7P2O15. (C23H26N2O4)4.

8 Jones, W., J. Biol. Chem., 1916, xxiv, p. iii.

Two structural formulas for a dinucleotide are given below, which differ from one another in only one respect. In I the two mononucleotide groups are joined to one another through their carbohydrate groups, and the formula represents a substituted disaccharide. In II the two mononucleotide groups are joined to one another through their phosphoric acid groups and the formula represents a substituted diphosphoric acid. HO\

HON

HO

HO\

0= P − .0 . CsHgOs . CH4N

Formula I can properly be assigned to a dinucleotide that forms a tetrabrucine salt, and is the most probable formula for the dinucleotide under discussion. Formula II cannot be assigned to a dinucleotide that forms a tetrabrucine salt and therefore cannot represent the dinucleotide under discussion.

The mode of nucleotide linkage in the dinucleotide must of course exist also in yeast nucleic acid so far as two of its nucleotide groups are concerned, but it does not necessarily follow that this same mode of nucleotide linkage maintains throughout the entire nucleic acid molecule. However, in the following article we shall give evidence to show that such is the case, and that the relation between yeast nucleic acid and adenine-uracil dinucleotide is that which is expressed in the formula.

EXPERIMENTAL PART.

Hydrolysis of Yeast Nucleic Acid with Ammonia at 115°, and Separation of the Products.

A solution of 100 gm. of commercial yeast nucleic acid in 530 cc. of 2.5 per cent ammonia was heated for 11⁄2 hours in an autoclave at 115°, and the cooled product was treated with 530 cc. of absolute alcohol. The bulky gelatinous precipitate thus formed was filtered on a Buchner funnel and drawn as completely as possible with a filter pump. The autoclave product is in this way separated into two fractions which may for convenience be designated as the guanine fraction and the adenine fraction. One fraction contains principally guanylic acid and forms an ammonium salt that is almost insoluble in 40 per cent alcohol. The other fraction is almost entirely adenine-uracil dinucleotide, whose ammonium salt is easily soluble in 60 per cent alcohol. When, therefore, a solution of the two fractions in ammonia is treated with an equal volume of alcohol, a sharp separation is effected.

The aqueous alcoholic solution of the dinucleotide was acidified with acetic acid, diluted with an equal volume of hot water, and treated with an aqueous solution of lead acetate as long as the reagent formed a precipitate with a portion of the cooled and filtered solution. About 450 cc. of 25 per cent lead acetate are required. When the precipitation was complete the solution was cooled and the heavy granular lead salt was filtered on a Buchner funnel and washed with a little cold water.

The lead salt was made into a thin paste with boiling water and decomposed with hydrogen sulfide. After the filtrate from lead sulfide had been carefully freed from every trace of hydrogen sulfide by boiling, it was evaporated at 50° under diminished pressure to about 50 cc., and treated with a large amount of absolute alcohol. The precipitated dinucleotide was hardened with absolute alcohol and dried with sulfuric acid in a vacuum desiccator. From 300 gm. of yeast nucleic acid 174 gm. of the crude dinucleotide were obtained.

Adenine-Uracil Dinucleotide.

Specimens of the dinucleotide prepared in the manner described always contain a small amount of guanylic acid and by acid hydrol

ysis produce a trace of guanine. The guanylic acid may be completely removed in the following way.

A solution of 100 gm. of the crude dinucleotide in 200 cc. of water is made alkaline with ammonia and treated with 300 cc. of absolute alcohol. The filtered solution is diluted with an equal volume of hot water, acidified with acetic acid, and precipitated with lead acetate as described, but the lead compound is washed with water, until every trace of soluble ammonium salt is removed. This can be done easily and without using the immense volume of wash water that would otherwise be necessary, by grinding up the lead precipitate to a thin paste with boiling water and drawing off the cooled liquid as completely as possible with a filter pump. After the washing in this way has been repeated two or three times, the wash water fails to give off ammonia with sodium carbonate in the cold.

The lead salt is decomposed with hydrogen sulfide, the filtrate from lead sulfide, after boiling until free from the gas, is evaporated, at 50° under diminished pressure, and the pale yellow syrup is treated with a large excess of alcohol. The precipitated dinucleotide is hardened and dried as described.

During the evaporation of the final aqueous solution a persistent bumping (due probably to the absence of ammonium salts) causes a serious loss of material so that the yield is lower than might have been expected. From 100 gm. of the crude dinucleotide 55 gm. of the purified product were obtained, or about 36 per cent of the commercial yeast nucleic acid used. The theoretical yield from pure dry nucleic acid is about 50 per cent. The dinucleotide is an amorphous white powder, soluble in water in all proportions, but insoluble in absolute alcohol. It responds to the color reactions for pentose with orcine and phloroglucine, and by acid hydrolysis yields an abundance of adenine but no trace of guanine.

A solution of 100 mg. of the substance in 2 cc. of 5 per cent sulfuric acid was heated for an hour at 100° and the hot solution was made alkaline with ammonia. No guanine was precipitated even after the solution had stood for a week, but upon the addition of ammoniacal silver nitrate, the fluid became solid with characteristic transparent gelatinous silver adenine. Tests for guanine and adenine made in this way never give misleading results.