March 20, 1960
COMMUNICATIONS TO THE EDITOR
1507
Whatever is the explanation, it seems that this is the first observed case of an association (other
in the general reaction a product identical with the aldahexoses and in the secondary reaction of hexoses than coagulation of electrically charged colloidaI gave a product identical with glucose. A solution particles) that requires an activation energy. of alkaline phosphatase containing 30 pg. N per DEPART~XENT OF CKEMISTRY K. s. DA4 ml. after 2 hr. a t 100’ with 1 N HC1 was found to contain 38 pg. glucose as determined by the reducSTATEUNIVERIITYCOLLEGEOF FORESTRY AT SYRACUSE UNIVERSITY M. FELD ing sugar method as determined by the cysteine SYRACUSE 10, NEW YoRK M. SZWARC reaction on the untreated material. The hydroRECEIVED JANUARY 19, 1960 lysate was found to contain approximately 25 pg. glucose as determined with glucose oxidase.1° The ninhydrin-phosphomolybdate positive GLUCOSE, A CONSTITUENT OF ALKALINE material was found to migrate as a cation a t PH PHOSPHATASE’ Sir: 8 and to be unstable in more alkaline solutions. Alkaline phosphatase of swine kidney, found in Other reactions of the compound were an immediate the ribonucleoprotein particles of the microsomes2 reaction with K3FeCNe and FeCl3 (blue) and an imand released as an active fragment by proteolysis,2r3 mediate reaction with K3FeCN6 alone (blue). has been isolated in an apparently homogeneous Both these reactions were destroyed by previous condition by application of the procedures pre- treatment with traces of cupric ion with exposure viously describedS plus ion-exchange chromatog- to air. In a study of model compounds, pkenolic raphy with Ecteola c e l l ~ l o s e . ~In a typical puri- compounds appear to have been eliminated but fication 2,000,000 units activity2 with a specific similar reactions have been observed with tri- and activity of over 100,000 units per mg. total N tetra-substituted pyrimidines; from the absorb(micro-Kjeldahl) was placed on a column of Ec- ancy in the ultraviolet, from studies of the model teola 5 X 100 cm. and, after thorough washing with pyrimidines and from analogy with the proposed water, was eluted with a gradient of barium acetate structure for vicine,’l i t is suspected that the matea t pH 9 varying from 0.01 to 0.05 iZI in 10 liters of rial may be a diamino-5-hydroxypyrimidine atsolution. The active material was eluted sym- tached, in the active material, to the glucose by a metrically near 0.03 M and was concentrated by glycosidic linkage a t the 5-hydroxy position. the barium procedure3 to yield about 1,500,000 (10) “Glucostat,” Worthington Biochemical Corp , Freehold, h-.J. (11) A. Bendich and G. C. Clements, Biochirn. Btophys. Acta, 12, units material with a specific activity of 295,000 to 462 (1953). 310,000 on the basis of total N. RechromatogDEPARTMEXT OF BIOCHEMISTRY raphy on Ecteola-cellulose, on Deae-cellulose4 FRANCIS BINKLEY or on D0wex-2~~ paper electrophoresis and paper EMORYUNIVERSITY ATLANTA 22, GEORGIA chromatography (ethanol-1 M ammonium aceRECETVED JANUARY 18, 1960 tate, 70-30) revealed no dissociation of absorbancy a t 280 rnp from activity. The material was free of peptidase and diesterase activity when tested unACID (CF,)?POH AND T H E diluted, amino acids were without effect on the T H E PHOSPHINOUS DIPHOSPHOXANE (CF,),POP(CF,,)zI activity and at no time was i t possible to demonstrate dialyzable cofactors other than magnesium Sir: We have recently isolated the new-type comion. The absorbancy in the ultraviolet was characas stable teristic of protein with a maximum a t 278 and a pounds (CF3)zPOH and (CF3)?POP(CF3)z minimum a t 250 mu. However, in the course of liquids contrasting with the apparently complete treatment with dilute acid (0.1 to 1.0 M a t 100”) instability of the corresponding hydrocarbon dethe absorbancy was found to increase remarkably rivatives. Evidently the highly electronegative and, a t the end of 2 hr., the absorbancy a t 278 was CF3 groups lower the power of phosphorus lonenearly tripled. There was a parallel release of pair electrons to bond either H + or (CF3)2P+cornreducing of ninhydrin reactive material,6 ing from 0. Thus these new (CF3)zPcompounds and of material reacting with pho~phomolybdate.~do not undergo the rearrangements RzPOH -+ H 0 Paper chromatography (propanol-water, 80-20) ~ probably separated a phosphomolybdate and ninhydrin reac- RzPOH and RzPOPRz + R z P - P R which tive material from a ninhydrin negative but aniline represent the first stages of decomposition when R hydrogen phthalate positive8 (brown color) mate- is a hydrocarbon group. Synthesis and Characterization of the Dirial with the same Rfas glucose. The untreated reaction 2(CF3)2PI Agzmaterial in the cysteine methods of Discheg gave phosphoxane.-The COS -+ COZ 2AgI (CF3)2POP(CF3)2 (room (1) These studies were supported by grants from t h e U. S. Public temperature, repeated shaking with fresh silver Health Service. Detailed studies of t h e purification, t h e effects of amlno acids and of divalent metal ions will be described b y C. Lea carbonate) gave yields above 79y0. The unused (2) F. Binkley, J. Davenport and F. Eastall, Biochem. Biophys. Re(CFJ2PI (1%) was converted by AgCl to the easily
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Corn.. . 1.. 206 (1959). . . (3) F. Binkley, V. Alexander, F. E . Bell and C. Lea, J . Bioi. Chem., 228. 559 (1957). (4) E. A. Peterson and H. A. Sober, THIS JOWRNAL, 78, 751 (1956). (5) N. Nelson, J . Bid. Chern., 163, 375 (1944). (6) S. Moore and W. H . Stein, ibid., 176, 367 (1948). ( 7 ) 0. Folin and V. Ciocalteu, ibid., 73, 627 (1927). (8) S. M. Partridge, Nalure, 164, 443 (1949). (9) Z. Dische, in D. Glick, “Methods of Biochemical Analysis,” Vol. 2 , Interscience Publishers, Inc., New York, N. Y., 1935, p. 313 8.
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(1) This research was supported b y t h e United States Air Force under Contract A F 33(616)-5435 (Subcontract No. 1) monitored b y the Materials Laboratory, Wright Air Development Center, WrightPatterson Air Force Base, Ohia. (2) G. M. Kosolapoff, “Organophosphorus Compounds,” John Wiley and Sons, Inc., New York, N. Y., 1950, p. 144. We also have found t h a t reactions expected t o form (CHs)xPOH give nearly quantitative yields of (CH3)sPH and (CH8)zPOOH; and attempts t o make (CHs)2POP(CH3)2also give products suzgesting dis.xoportionation.
