rnn
Feb., 1942 rated. The residue wag crystallized from methanol. m. p. 17%180°. yield 1.5g. A d . Calcd. for Cdi,,O,: C. 73.7; H. 9.2. Found: C. 73.8; H. 9.1. The above material. MM mg.. wag hydrolyzed by boiling with 250 mg. of d i m hydroxide in 50 ff. or ethanol. The neutral fraction melted at 223-225' after crystallization from aqueous methanol. Am!. Calcd. for CIHnO.: C. 75.8: H 9.7. Found: C. 75.5; H. 9.6. e d hot The hydrolysis product muld he ~ ~ t y l a t with acetic anhydride 10 give (he o r i g i d acetate melting at 179-180'. Treatment of A1Wo-Pre~enol-3(~)0 Acetate rlth Hydrogen Peroxlde.-A mixture of 5 g. of A'*& pregnenol-3(B)-one-20 acetate. 250 m. of acetic acid. and €4ff. of 30% hydrogen peroxide WPI heated to 70' for five hours. The reaction mixture wag worked up PI d k b e d for pregnenolone acetate. The neutral mat& melted at 1SE-186° after crystallization from methanol. yield 2.5 g. Anal. Calcd. for G,IiaO,; C. 73.7; H. 9.2. Found: C, 73.8; H. 9.0. Hydrolysis of this material with alcoholic sodium hydroxide gave a product which melted at 181-182' nfts crystallization from methanol. This substanrr gave a large dep-on in melting point when mixed with the tate. m. p. 185-186°. A d . Calcd. for GtHaOl: C. 75.8; H. 9.7. Found: C, 75.5; H. 9.6. Reaeetylation with hot acetic anhydride save the origi. apI acetate melting at 18S186°.
Fie. I.--Avidin cryaia1.i rnawifinl IlHJIl
liiiirs
tallized repeatedly by the same method and retained high activity. For assay purposes, saltfree avidin was obtained by dissolving the crystallized material in dilute salt solution, and dialyzing the solution against distilled water until the avidin was completely precipitated. The crystallization procedure was somewhat destructive to the activity of the avidin. The potency of three times recrystallized material was approximately 4OOO units per gram, while the most active amorphous material previously obtained had a potency of approximately 7000 units per gram. Crystals which stood in the refrigerator &lOOL OF ~ E M l S T R KAND P€NSICS in contact with the mother liquor lost approxiTHE PENNSYLVANIA %ATE COLLEGE STATE COLLBDE.PENNA. RECENW A m a I. 1941 mately three-fourths of their activity in three weeks. Dry crystals which stood for three months a t summer temperature lost no activity. Crystalline Avidin Analyses on two independent batches of crysBV DERROL PFN"NDION. ESMOND E. SNBLL AND ROSER, tallized avidin gave the following results: E. EAKIN
Using essentially the procedures described in E previous communication for obtaining concen. trated material,' we have obtained the biotin-inac tivating protein, avidin, in crystalline form Highly concentrated material obtained by this means was dissolved (50 mg. per cc.) in one-half saturated ammonium sulfate solution, at m n i temperature, and saturated ammonium sulfate solution was added to inapient precipitation. After centrifuging out this first turbidity, the clear solution was placed in a refrigerator. Within an hour, the avidin precipitated in the form of fine needle-like crystals. When care was taken that the crystallization proceeded more slowly, larger plates (Fig. 1) were obtained. Both forms were equally active. Such material was recrys(I) &kin.
Smell and Willimnu. 1. Bid.
Ch-.. 140. b9.S--51.7
Sample1 Sample2
C
H
N
S
Rsihe
43.72 14.2(1
7.60
12.10 L2.M
1.32
2.09
... 0.7.5 Both samples gave a positive Molisch test for carbohydrate, and the analyses indicate that the substance may be a protein with a large carbohydrate moiety. Further study of the substance will await its production on a large scale. i.2X
DEPARTMENT OF CHEMISTRY
TEXAS AUSTIN. THXAS UNIVERSITY OF
RECEIVED OCTOBER11. I941
An Improved Method for the Preparation of Benzenediazonium Salts BY WILLIAMSMITH AND CHAS.E. WARING
The usual method of preparing diazo salts is that give11 by Hantzsch and Jocheni.' Essen-
