Dec. 20, 1952
COMMUNICATIONS TO THE EDITOR
atives of 11. Furthermore, the infrared spectra (Nujol mulls) of closely related acyl derivatives of I1 exhibit differentiating absorption in the 12 to 14 micron region. Thiolutin is characterized by two similar, sharp bands a t 12.1 and 12.5 microns, a dominantly, strong band a t 13.45 microns and a partially resolved band a t 13.6 microns. Aureothricin exhibits two similar, sharp bands a t 12.2 and 12.6 microns, a partially resolved band a t 13.45 microns and a dominantly strong band a t 13.6 microns. Structural studies are in progress and will be reported in subsequent publications.
6305
C-TERMINAL GROUPS OF TRYPSINOGEN, DFPTRYPSIN AND CARBOXYPEPTIDASE1
Sir:
A recent study of the effect of carboxypeptidase on chymotrypsinogen and DFP-a-chymotrypsin has led to the conclusion that the zymogen contains no C-terminal groups, in contrast to two such groups, leucine and tyrosine, in DFP-a-chymotryps h 2 In the present study, the same experimental technique was employed to investigate similarly the changes involved in the activation of trypsinogen and to test for the autolysis of carboxypeptidase. WALTER D.CELMER Trypsinogen was crystallized in the presence of 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 6, NEWYORE: BROOKLYN 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 DFP 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 it 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. BADER~ (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 to Professor Desnuelle for informing Milwaukee, Wisconsin. us of these reiults prior to publication.
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