April 5, 1956
PEPTIDEDERIVATIVES CONTAINING HYDROXYAMINO ACIDS
TABLE IV TOTAL ACTIVITYAND SPECIFIC ACTIVITYOF FRACTIONS OBTAINED AT VARIOUSSTAGES OF THE PURIFICATION OF DAMINOACIDOXIDASE Specific activity, PI.
Stage of purification
Total actfvity, PI. 01/10 min.
Ot/lO
min./mg. protein
Crude extracts 764,000" 6.4 Ppt. I First ammonium sulfate 1,200,000 640 PPt. Ppt. I1 Second ammonium sulfate ppt. 1,010,Ooo 1390 This figure must be doubled due to the presence of catalase in the crude extract. Catalase is not present in subsequent fractions. monium sulfate t o 0.4 and the PH t o 7.0. The white precipitate which formed was collected by filtration after first adding 10 g. of Hyflo Super-Cel filter aid. The "Ce1"-enzyme filter cake was extracted four times with 130-ml. portions of M / 2 5 pyrophosphate buffer, PH 8.3, t o give a final volume of 530 ml. of a clear yellow solution. This solution was brought t o 0.3 saturation with respect to ammonium sulfate and the yellow precipitate which formed was collected by centrifugation a t 317 X gravity for 20 min. The yellow precipitate was resuspended in distilled water t o yield 100 ml. of dark amber solution. Data on the activity of the various fractions obtained in the course of this procedure are presented in Table IV. Specific Property Test .-The procedure of Falconer and Taylor6 was used. A graphic representation of the results is presented in Fig. 4 . Column Chromatography.-Hyflo Super-Cel was treated according t o the Drocedure of Clauser.'* Five grams of this treated material was added, as a slurry with th; buffer (pyrophosphate pH 6.0) in which absorption was to take place, t o a 7 X 350 mm. column. The column was washed with more of the same buffer and then 3.0 ml. of the enzyme con(12) H. Clauser and Choh Hao Li, THIS JOURNAL, 76, 4337 (1954).
[CONTRIBUTION FROM
THE
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taining 10 mg. of protein in buffer p H 6.0, added. The enzyme was eluted from the column by slowly raising the pH of the column. A continuous PH gradient was maintained along the column by allowing a more alkaline pyrophosphate buffer, p H 8.3, to siphon into a well stirred acidic pyrophosphate buffer, pH 6.0, which in turn siphoned into the column. The rate of elution was adjusted t o 5 ml./hr. An automatic fraction collector was used t o collect the fractions. The whole procedure was conducted in a cold room a t 0". All of the protein (10 mg.) was recovered in 100 ml. of eluate. Filter Paper Electrophoresis .-The apparatus was of the hang-strip modification built after the design of Kunkel . I 3 The purified enzyme solution was added from a 15 lambda micropipet t o inch wide strips of Whatman No. I1 paper. The application was made at the apex of the strip after dialyzing the enzyme against M / 2 0 alanine buffer, $H 8.3 for four hours. A small drop of dextran was also placed a t the apex in order t o account for electroiismosis. Applied potentials ranged from 200 t o 500 volts and times from 2-12 hours. At the completion of an experiment the position of the protein band was determined by staining with brom phenol blue in the manner described by Durrum.l* In addition several of these strips were segmented immediately after electrophoresis and activity determinations made on each segment. The position of maximum activity always corresponded to the position of the protein band within the limits of experimental error. The results of such an experiment are presented in Fig. 3. Conventional Electrophoresis.-Purified enzyme solutions were first dialyzed overnight against the buffer in which the electrophoresis was to take place. Protein determinations made before and after dialysis were the same within experimental error. The positions of the boundaries were recorded photographically a t appropriate intervals. F r k tions were withdrawn from the cell a t the end of the experiment by means of a syringe and needle. The activity and protein content of these fractions were determined in the usual manner. The results of these experiments are presented in Fig. 4 and Table 111. (13) H. G. Kunkel and A. liselius, J. Gen. P h y s i o i . , 35, 89 (IQRI). (14) E. I. Durrum, THISJ O U R N A L , 7 9 , 2043 (1950).
COLLEGE PARK,MARYLAND
DEPARTMENT OF CHEMISTRY, MASSACIIUSETTS INSTITUTE
OF
TECIfsoLOG~]
Peptide Derivatives Containing Hydroxyamino Acids BY JOHN C. SHEEHAN, MURRAY GOODMAN' AND GEORGE P. HESS* RECEIVED SEPTEMBER 12, 1955 With the use of N,N'-dicyclohexylcarbodiimide as the condensing agent, several peptide derivatives containing L-serine, L-threonine and L-hydroxyproline have been synthesized
I n view of the outstanding importance of proteins and peptides containing hydroxyamino acand the complex problems associated with their synthesis, we have extended our recently devised method of peptide formation9 to include derivatives of serine, threonine and hydroxypro(1) Aided by a contract from the Office of Naval Research. (2) Aided by a fellowship from the National Foundation for Infantile Paralysis. (3) N. K. Schaffer, S. C. May, Jr., and W. H. Summerson, J. B i d .
Chem., 202, 67 (1953). (4) N. K. Schaffer, S. Harshman, R. R. Engle and R . W. Drisco. Fed. Proc., 14, 275 (1955). (5) M. Flavin, J. B i d . Chem., 210, 771 (1954). (6) F. Sanner and H. Tuppy, Biochem. J.,49,463, 481 (1951). (7) P. H . Bell, e2 nl., THISJOURNAL, 76, 5565 (1954); P . H. Bell, et ol., ibid., 7 7 , 3419 (1955). (8) T . Wieland and W. Schon, Ann., 693, 157 (1955). (9) J. C . Sheehan and G. P. Hess, THISJOURNAL, 77, 1067 (1955).
line. Difficulties previously encountered have involved mainly interference by the hydroxyl group. Successful syntheses of these compounds have been accomplished only via the azide method, a multistep procedure.1°-14 With our technique, N,N'dicyclohexylcarbodiimide'5 was used to form the peptide bond. Although the first aliphatic carbodiimide, diethyl carbodiimide, was prepared as early as 1893,'O the reactions of these compounds with (10) J. S. Fruton, J. B i d . Chem., 146, 463 (1942). (11) D.W. Woolley, ibid., 172, 71 (1948). (12) K. Hofmann and A. Johl, THIS JOURSAL, 77, 2914 ( 1 9 3 3 ) . (13) E. L. Smith and M . Bergmann, J . B i d . Chem., 153, 627 (1941). (14) M.Brenner, K.Riifenacht and E. Sailer, H e l v . C h i m . A c l a , 3 4 , 2096 (1951). (15) In reference 9 the wrong authors unfortunately were cre(litc(1 for the preparation of this reagent. The correct authors are E . Srhrnl
