Racemic (dl)-hydrocarbon prepared in the same way had b.p. 168-170", E ~ ~1.4130, D m.p. -26.5', and the same iiifrared spectrum.
or 0.05 AUbuffers, the change in optical density with t h e a t 302 inp is shown in Fig. 1, I. The first-order reaction taking place a t pH values above Acknowledgment.-\Ye should like to thank Pro- neutrality is probably a hydrolysis of the imide fessor !Ar. E. Doering for originally suggesting this linkage since the resulting product combines with research and Sigma Xi for a grant to one of US mercaptans only very slowly and has an absorption (T. R. T.). spectrum indistinguishable from that of synI ~ E P A R T M E S OF T CHEMISTRY A C D CHEMICAL ESC~SEERISG thetic N-ethylmaleaniic acid. This instability ~ V I V E R S I T YOF CAL~PORNIA should be taken into account in any quantitative BERKELEY 4, CALIPORWA procedure using S E X , such as the titration of sulfhydryl groups,? i n which there is any possibility The Stability of N-Ethylmaleimide and its Reac- of the reagent being exposed to alkaline conditions. The rate of reaction of X E l l with GSH as a tion with Sulfhydryl Groups functio~iof p H is shown in Fig. 1, 11. Clearly, at B Y JOHC D G R E C O R ~ about neutrality, the reaction with GSH is so much [aster thaii the decolilposition of the reagent that RECEIVEI> FEBRL A R Y lti, 1933 it may be used for quantitative purposes. The I\--Ethylmaleimide ( N E l I j has been shown to absolute chanre in molar extinction coefficient react rapidly and specifically with sulfhydryl ( A E M 0.61 X lo3), however, is only one-twelfth groups,' to be useful in the titration of such groups of that observed by Boyer4 a t 250 inp when pi n proteins,a and to be an antimitotic agent.' In niercuribenzoate reacts with mercaptans, ( A E M view of its potential application to the studv of 7.6 X lo3),and in general it is probably not sensibiochemical reactions involving sulfhydryl groups, tive enough for photometric titration of protein me have made some observations on the rate of sulfhydryl groups. reaction and the stability of NEM. On the basis of this optical method, there is no Changes in the absorption spectrum of N E X 011 evidence of appreciable reaction of X E X a t pH 7 reaction with glutathione (GSH), in the region of with S-acetyl GSH, diacetyl 2-niercaptoethyla205-240 mw, have been observed by Friedmann. mine, oxidized glutathione, ethanol, ethylamine or He shows also the broad peak centered a t 302 nip HCN. Cysteine and H,S react rapidly, as does (molar extinction coefficient, E M F20), but does not potassium borohydride a t p H 9. For comparison, inention its behavior in this reaction. The vir- some determinations have been made of the rate of tually complete disappearance of this peak, which combination of NEM with myokinase as assessed takes place on combination with mercaptans, or by inactivation of the enzyme. -It pH 7.,5 maxion decomposition of NELI, has been used here to mal inactivation (!?>yo) was not reached until after follow the rate of reaction and the instability of the treatment for 30 minutes with 2 X IIf KEN. compound a t various PH values. Excess XEM must have been present, since a t For 1 2.5 X 1 0 P 3 JP solutions of ?\'EM in ~ + a t e r /iH 9 as little as 3 x 10 - 2 -I/ NE11 gave thc S ~ I I I C inactivation in 3 Ininutcs. ' ~ ' h ctiiile cLir\-e of i i i activation was sinooth u i i d show-ctI 110 evideiicc ior classes of sulfhydryl groulis liaving differelit rc~tivities.~ The situation with this enzyme is not clear, however, since the original preparation was activated fivefold when assayed in the presence of GSH; yet in the original state it was susceptible to almost complete inactivation by S E X . The GSH activation is thus not explainable simply by the existence of sulhydryl groups as disulfides, in which condition they should be iinrnuiie to the inhibitor. 7
1
-
_
-ItTI---T-T----, 1
Experimental Spectra were obtained in a Car? Recording Spectrophiltometer, and reactions were followed it1 a Beckman Model (! 2 4 6 2 2 0 1 2 3 DC Spectrophotometer. I, hours. 11, minutes:. S-Ethylmaleamic acid was synthesized from maleic anhydride and ethylamine as described by Piutti.6 Fig 1,-1, rate of decomposition of 1.25 X li!-3 .lI Myokinase was prepared from horse muscle, essentially S E M in water or 0.05 JI potassium acetate, pH .5.0 ( A ) ; by the procedure of Colowick and Kalckar' with the addipotassium phosphate, PH 7.0 (B) ; tris-(hydroxymethyl) - tion of a fractionation between 17.5 and 35% saturated aminomethane, pH 8.0 (C) ; Z-amino-2-methyl-I,3-propane- ammonium sulfate. The enzyme was assayed in a systcill containing 200 p M tris-(hydroxymethy1)-aminomethane diol, pH 9.0 ( D ) . 11, rate of reaction of SEiM with GSH buffer, p H 7 . 5 , 5 p.W magnesium chloride, 2.5 p J I adenosine (both 1 0 F 3 M) in 0.1 -11 sodium acetate, pH 5.0 ( A ) ; potasdiphosphate, 20 p J l GSH, 30 p-M glucose, and excess hexosium phosphate, PH 0.1 ( H ) , and PH 7.0 ( C ) . kinase in 1.0 ml., b?-measuring loss of acid-labile phosphate. Of the above prep:rration, 3 pg. of protein tr;in\ferred 1 .O
( 7 ) S I' (1'14'18
i'ciluwick an.i I I \ I
l < : ~ l r k ~ r.r., Iiijl
(
h c w , 148, I I i
3923
July 20, 1955
NOTES
Inactivation was carp M phosphate in 10 minutes a t 30'. ried out by incubating solutions containing 0.25 mg. per ml. protein and 2 X 10-4 M NEM a t room temperature, and diluting with a large excess of GSH a t appropriate time intervals. This work was supported by a research grant from the Cancer Institute of the National Institutes of Health, Public Health Service. BIOCHEMICAL RESEARCH LABORATORY MASSACHUSETTS GENERAL HOSPITAL BOSTON14, MASSACHUSETTS
be made available. With two small laboratory generators, sufficient phosphine was made in 30 hours to make over 1100 g. of THPC. In this case the phosphine produced by 30 g. of aluminum phosphide was absorbed and allowed to react with 100 g. of formaldehyde-hydrochloric acid solution in less than one hour. A good yield of T H P C and an easily isolated product is obtained when about 4.2 moles of formaldehyde are used per mole of hydrochloric acid. Paraformaldehyde or polyoxymethylene may be substituted for the formaldehyde without affecting the rate of the reaction or yield of product. The phosphine used may be made by any method desired. Relatively pure phosphine is obtained conveniently by treating aluminum phosphide with water a t room temperature. This source supplies a steady stream of the gas. -4luminum phosphide is not generally available, but i t can be made by igniting with adequate precautions a mixture of 1.5 to 2.0 parts (by weight) of powdered aluminum and one part of red phosphorus.
~
~~
~
Laboratory Preparation of Tetrakis-(hydroxymethyl)-phosphonium Chloride BY WILSONA. REEVES,FRANCIS F. F L Y S NASD ~ GUTHRIE RECEIVED FEBRUARY 7, 1955
JOHN
D.
Tetrakis-(hydroxymethy1)-phosphonium chloride, (HOCHZ)dPCl, which is made by treating phosphine with an aqueous solution of formaldehydehydrochloric acid. recently has attracted interest as an ingredient of certain resins which impart flameresistance to cotton fabrics.?",b The only availExperimental able method for making this compound was reA . Apparatus.-The apparatus consisted of two phosported by Hoffman in 19?l.3 His method is slow phine generators provided with safety water-seal pressure and somewhat hazardous, since frequent minor ex- releases, and a reaction vessel. Each generator conplosions occur during the reaction. Since it ap- sisted of a two-liter filter flask fitted with a two-hole rubber peared that tetrakis- (hydroxymethyl)-phosphon- stopper. Through one of the holes was a n inlet tube used t o admit nitrogen and the other was connected to a safety ium chloride, abbreviated T H P C for convenience, water-seal pressure release. The water-seal pressure rewould be in demand for laboratory and perhaps lease could be of any convenient design as long as the water commercial use, improvements were required in levels in it can be varied from 0 to about 15 inches. The the details of its preparation. The purpose of this side arm of each filter flask was connected t o the reaction vessel. The reaction vessel was a five-liter jar fitted with a report is to present a practical laboratory method four-hole wooden cover. The inlet tubes, which consisted for preparing THPC. of gas dispersion filter tubes, passed through two of the Heretofore, a temperature of SO" was considered holes and extended nearly t o the bottom of the vessel. A most desirable and was used for treating phosphine high speed stirrer and a vent tube were mounted through with an aqueous solution of formaldehyde and hy- the other two holes. B. Reagents.-( 1) Nitrogen. (2) Formaldehl-de-hydrochloric a c i d 3 -It lower temperatures the reac- drochloric acid solution was prepared by mixing 37% tion was thought to be slow and a t higher tempera- formaldehyde and 35% hydrochloric acid so that the final tures the vapor pressure of the solution interfered solution contained these reagents in about a 4 . 2 : l mole with the absorption of phosphine. Since phosphine ratio, respectively. (3) Aluminum phosphide: about 100g. portions of a mixture of 528 g. of powdered aluminum is spontaneously inflammable a t SO", frequent ex- and 352 g. of red phosphorus were placed on an asbestos plosions occurred in the reaction vessel of the Hoff- mat in a fume hood and ignited with a match. The comman method. By that method it required about 8 bined portions made about 880 g. of crude aluminum phoshours for the phosphine generated from 30 g. of alu- phide. Small portions of the aluminum-phosphorus mixture were ignited because intense heat and toxic vapors are minum phosphide to be absorbed and to react with produced when these elements are burned. (4) Phosphine 100 g. of formaldehyde-hydrochloric acid solution. was generated by adding aluminum phosphide to water in We have found t h a t phosphine will react rapidly the apparatus described above. C. Procedure.-All operations were performed in a well with formaldehyde-hydrochloric acid solutions a t ventilated hood because of the toxicity of phosphine. room temperature. Phosphine is more soluble in About 1600 ml. of water was added to each phosphine these solutions a t 25" than a t 80", and a t the lower generator, then water was added t o each safety watertemperature explosions are eliminated. If the re- seal pressure release tube. Formaldehydehydrochloric acid action is carried out a t 50°, explosions occur only solution, made by combining 660 g. of 35% aqueous hydroacid and 2100 g. of 37% aqueous formaldehyde, was occasionally. However, since heating is not nec- chloric put into the reaction vessel. The stirrer in the reaction essary for the reaction, a temperature of about 25" vessel was adjusted for vigorous agitation and then the is preferred; temperatures of 10 to 15' also are sat- apparatus was swept out with nitrogen by allowing a steady isfactory. It appears that the primary factor stream of the nitrogen to pass through it for about 10 At the time the nitrogen was stopped, about 5 g. which controls the amount of T H P C that can be minutes. of aluminum phosphide was added t o each generator. After made in the laboratory from a specified amount of about one hour, 15 g. of aluminum phosphide was added to formaldehyde-hydrochloric acid solution in a given each generator and so on at hourly intervals until the 880 g. length of time is the amount of phosphine that can had been added. Two hours after the last addition of (1) Deceased. (2) (a) W. A. Reeves and J. D. Guthrie, U. S. Department of Agriculture, Agricultural and Industrial Chemistry Bulletin, AIC-364 (1953); (b) W. A. Reeves and J. D. Guthrie. Textile W o r l d , 104, 101 (1954). (3) A . Hoffman, THIS JOWRNAL, 43, 1684 (1921).
aluminum phosphide the reaction vessel was disconnected and the solution was transferred t o large evaporating dishes; The volatile components were evaporated a t about 70 to 75 with stirring until crystals began to form, when the mass was transferred t o a desiccator and allowed t o cool to room temperature over sodium hydroxide pellets. The entire mass crystallized. Approximately 1135 g. of 95% T H P C
