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atives of 11. Furthermore, the infrared spectra C-TERMINAL GROUPS OF TRYPSINOGEN, DFPTRYPSIN AND CARBOXYPEPTIDASE1 (Nujol mulls) of closely related acyl derivatives of I1 exhibit differentiating absorption in the 12 to 14 Sir: micron region. Thiolutin is characterized by two A recent study of the effect of carboxypeptidase similar, sharp bands a t 12.1 and 12.5 microns, a on chymotrypsinogen and DFP-a-chymotrypsin dominantly, strong band a t 13.45 microns and a has led to the conclusion that the zymogen contains partially resolved band a t 13.6 microns. Aureo- no C-terminal groups, in contrast to two such thricin exhibits two similar, sharp bands a t 12.2 groups, leucine and tyrosine, in DFP-a-chymotrypand 12.6 microns, a partially resolved band a t 13.45 sin.2 In the present study, the same experimental microns and a dominantly strong band a t 13.6 mi- technique was employed to investigate similarly crons. the changes involved in the activation of trypsinoStructural studies are in progress and will be re- gen and to test for the autolysis of carboxypeptiported in subsequent publications. dase. Trypsinogen was crystallized in the presence of WALTERD.CELMER RESEARCH LABORATORIES FREDW. TANNER,JR. DFP3 and residual trypsin (less than O . O 1 ~ o ) was CHAS. PFIZER AND CO., INC. M. HARFENIST inactivated by the addition of a 2-fold excess of BROOKLYN 6, NEWYORE: T. M. LEES I. A. SOLOMONScrystalline soybean trypsin inhibitor (Worthington). Twice recrystallized D F P - t r y p ~ i ncontain,~ RECEIVEDNOVEMBER 10, 1952 ing less than 0.1% trypsin, was similarly inactivated. Seven times recrystallized carboxypeptidase was freed from residual amino acids (which previTHE REACTION OF DIKETENE WITH KETONES ously were found to form a background on paper Sir : chromatograms2) by washing of the crystals with Although the preparation of diketene in acetone distilled water, and residual tryptic activity (less is a commercial process, its reactions with ketones than o.0470) was removed by the addition of a 100to form compounds formulated as 2,2-disubstituted- fold molar excess of D F P to a solution of the en4-methyl-6-keto-l,S-dioxenes, I, have escaped ob- zyme in 10% LiC1. Trypsinogen or DFP-trypsin was incubated with servation. carboxypeptidase (substratelenzyme mole ratio 0 11/1) a t PH 7.5,2501and aliquots were removed a t 'I various time intervals up to 3 hours. Any free amino acids were absorbed onto and eluted from Dowex 50 resin2j4and subjected to one-dimensional paper chromatography (butanolacetic acid-water, 4: 1:5 or phenolm-cresol, 1: I). No amino acids The p-toluenesulfonic acid catalyzed reaction of whatsoever could be detected. Negative results diketene with acetone a t 90' yields Ia (91%), were obtained also when a 2% solution of carboxyb.p. 61-84' (5 mm.), n Z o1.464, ~ ( P O 4 1.079, A,,,, EtOH peptidase was similarly tested after i t was al247.5 mp, log E 3.9; Anal. Calcd. for C'IH1003: lowed to autolyze up to 3 hours a t 25", pH 7.5. These negative results suggest that (I) the proC, 59.14; H, 7.09; Found: C, 59.20; H, 7.15. The product from acetophenone, Ib, is crystalline, tein substrates have no free C-terminal groups or m.p. 93-94O, A",t,",H 247.5 mp, log E 3.86, A,,,,isooctane (2) that these groups are not reactive toward car240 mp, log E 3.85; Anal. Calcd. for Cl2H12O3: C, boxypeptidase either because they do not conform 70.57, H, 5.92; Found, C, 70.21; H , 5.81. These to the specificity requirements of the enzyme or ketodioxenes are pleasant smelling liquids or crys- because they are sterically inaccessible. The first talline solids easily handled in the absence of alkali. explanation is rendered unlikely by the recent findAs many of their reactions parallel those of diketene ings of Rovery, Fabre and Desnuelle5 that trypsinothey may conveniently be used in its place. Thus I gen and DFP-trypsin each contain one N-terminal reacts with alcohols, aniline and with mild alkali to group, valine and isoleucine, respectively, suggestyield acetoacetates, acetoacetanilide and dehydra- ing that these proteins are composed of a t least one cetic acid, respectively. I does not react with car- open polypeptide chain. The second interpretabonyl reagents and this and the ultraviolet spectra tion receives support from the observation that following denaturation by acid, DFP-trypsin becomes rule out structures I1 and I11 also considered. reactive toward carboxypeptidase (substrate/enOH
(I) DFP denotes di-isopropylfluorophosphate. This work was performed under contract No. Nonr-477-04 between the University of Washington and the Office of Naval Research, Department of the ACII. Navy, and was supported also by funds made available by the people CHXOCH~COOCH of the State of Washington, Initiative 171. R ', (2) J. A. Gladner and H . Neurath, Biochim. ct Biophys. A d a , 9,335 I1 R~ R~ (19521, and unpublished experiments. RESEARCH LABORATORIES (3) L. W. Cunningham, Jr., F. Tietze, N. M. Green and H. Neurath, A. BOAKE,ROBERTS AND Co. LTD. MICHAEL F. CARROLL Discussions of the Faraday Society, in press; F . Tietze, in preparation. E. 15, ENGLAND LONDON, ALFREDR. B A D E R ~ (4) S. M. Partridge, Nature, 169, 496 (1952); A. R.Thompson, i b i d . , 169, 495 (1952). RECEIVED NOVEMBER 20, 1952 (6) M. Rovery, C. Fabre and P. Desnuelle, Biochim. et Biophys. (1) The Research Laboratories, The Pittsburgh Plate Glass Co., Acta, in press. We are indebted t o Professor Desnuelle for informing Milwaukee, Wisconsin. us of these reiults prior to publication.
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amply sufficient to permit ionic oxidation-reduction and metathetic reactions in dimethylformamide. Thus dichromate ion is reduced by dimethyldichlorosilane in this solvent to several lower oxidation states of chromium; ammonium thiocyanate undergoes metathetic reaction with the same chlorosilane to precipitate aminonium chloride; boric acid reacts to Eorm a silicone polymer of the "bouncing putty" variety; phosphotungstic acid precipitates a dimethylsilyl phosphotungstate; parunitrobenzoic acid dissolves in a solution of dimethyldichlorosilane in dimethylformarnide, but not in the amide itself nor in a mixture of the amide with hydrogen chloride. I n similar vein, we have found that some organogermanium halides form conducting solutions in DEPARTMENT OF BIOCHEMISfRY EARL\Y.DAVIE dimethylformamide and undergo ionic metatheses. USIVERSITY OF WASIIINGTOS SS 4TTLE, \vASIfINGTON ~ I A N SXEURATH While i t may prove possible to employ concentrated R a c b r v ~ uSOVEXBER 24, 19r12 hydrochloric acid as a dissociating solvent for organogermanium compounds because of an observable reversibility,lO the preparative utility of such a IONIZATION OF ORGANOMETALLIC HALIDES solvent is more limited. However, we have found Sir: that dimethyltin dichloride may readily be handled ,iltliough trimethyltin iodide' and the triethyl- in acidic aqueous solution; it hydrolyzes in pure lead halides? liave long been known to be soluble in water only to the extent of 10.5% in 0.064 molal water and to undergo met athetic reactions in q u e - concentration a t 25". Cryoscopic measurements in c)u? solution, the rapid and complete hydrolysis of water give a van? Hoff i factor of 2.60 a t 0.1693 organosilicon halides? precludes such reactions. molal concentration, 2.69 at 0.1041 molal, and The similar alcoholysis4 of organosilicon halides, 2.86 a t 0.0639 molal. The (CW3)Sn++ ions may and their related reaction with ammonia6 and am- be retained on a cation exchange resin and eluted ines6 prevent the use of these polar liquids as ioniz- with various acids. By elution and by metathesis ing solvents. we have prepared dimethyltin tungstate, molybFor much the same reasons, the electrochemistry date, sulfide, oxalate, succinate, naphthionate, salof organometallic compounds has been concerned icylate, phthalate, benzoate, ferricyanide, ferrocychiefly with the electrolytic behavior of organo- anide, iodate, arsenate, vanadate, cyanate and antimercury compounds in water,' the conductivity of rnonate. A report on the preparation! purification, trimethyltin iodide in acetone, alcohols. and pyri- and properties of these compounds is being predine,8 and of Grignard reagentsg in ether and re- pared. lated solvents. The organosilicon halides exhibit The financial assistance of the Mallinckrodt Fund 110 conductivity in ether. and the Office of Naval Research is greatly appreIVe now find that a considerable number of ciated. methyl-, ethyl-, dodecyl-, octadecyl- and phenyl (10) 13. G. Rochow, i b i d , 70, 1801 (1948). chlorosilanes are soluble in anhydrous dimethylKURTGINGOLD formamide, and that such solutions are highly conLABORATORY EUGENE G. ROCHOW ducting. For example, dimethyldichlorosilane ex- MALLINCKRODT HARVARD UNIVERSITY D ~ T M ASEYPERTH R hibits an equivalent conductance of 18 ohms-' a t a CAMBRIDGE 38, MASS. ALBERTC. SMITH,JR. concentration of 0.004 equivalent per liter, and 0.5 ROBERT WEST o1im-l a t one equivalent per liter, both a t 30". RECEIVEDOCTOBER 27, 1952 The conductances of eleven organochlorosilanes are being studied, and from these and the colligative properties of the solutions the degrees of ionic dis- ISOTOPE EFFECTS IN THE IONIZATION OF ALKYL CHLOROSULFITES sociation are being sought. Preliminary results indicate that the dissociation constants are rela- Sir : to lo-*. Examples occur frequently in the literature of tively small, in the range The chemical significance of these results lies in marked changes in the rate of reactions which the fact that the concentrations of organosilicon break bonds to hydrogen, when deuterium or triions of the types R a i f , RgSi+f, and RSi+++ are tium is substituted for this hydrogen. No appreciable effects have heretofore been observed when ( 1 ) G. Bredig, 2. physik. Ckem.,13, 303 (1894); N. Zelinsky and J. Krupiwin, i b d , 21, 47 (1896). the reactant and product have all bonds to hydro( 2 ) TV Klemm, FIAT Report of Inorganic Chemistry, Part 11. p. 189 gen intact. Such studies have in fact become a (1948). standard tool for the detection of bond-breaking in (3) W. I. Patnode and D. F. Wilcock. Tars JOURNAL, 68,358 (1946). the rate determining steps of reactions.le2 I n a n (4) C. A. Burkhard, J . Org. Chem., 16, 106 (1950). (5) R. 0. Sauer and R. Hasek, THISJOIJXNAL, 68, 241 (1946) experiment designed to study by this technique the (6) Y P; Voinov and A Reutt, J. Gen. Chcm. ( U S S R ), 10, 1600 mechanism of the elimination reaction which ac(1540)
zyme mole ratio l O / l ) , yielding lysine, and to a lesser extent leucine, incthioninc, glutamic and aspartic acids, threonine, serine and glycine. Denaturation was effected by 48 hours of exposure to HCI, pH 1, a t O", followed by 24 hoiirsof dialysis against HCl, pTI S. The present results are corllpatiblc with the suggestion that activation of trypsinogen is accoinpanied by the splitting of a peptide froin the aniino end of the polypeptide chain5 but it remains to be seen whether in analogy with the activation of chymotrypsinogen to a-chymotrypsin,? a peptide is split fro:ii the carboxyl end of the chain as well. Further quantitative studies of these phenomena are in progress and will be published a t a later time.
(7) F . Hein and H . Meininger, 2. srrorr. Chem., 146, 95 (1925). (8) C. A Kraus and C. C. Callis, TEISJOURNAL, 46, 2624 (1923) W E v a n s .ta-l F t c c ,bid 66. 1474 (1833) 8 6 , G5.l (1934)
(1) I'
1% Westheirner and N. N?colnides, THIS JOURNAL, 71, 25
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