4194
NOTES
VOl. 76
nine, aspartic acid and glutamic acid, some of them as more than one residue. On treatment with carboxypeptidase, only leucine is released from this peptide. Assuming the prior removal of Glu.Phe from the C-terminus, this result is consistent with the C-terminal sequence: . . . . Pro,Leu.Glu.Phe.8 Thus, it appears that chymotrypsin splits the Cterminal sequence between leucine and glutamic acid. The splitting of this bond and the failure to split a t a second phenylalanine residue farther down the chain appear to be deviations from classical conc e p t ~of~ chymotryptic specificity. However, the final answers to these and otherlo apparent anomalies of enzymatic activity must await the careful study of a wide range of synthetic peptides with highly purified enzymes. Work is in progress to locate the exact position in corticotropin-A of the arginyltryptophan sequence. Present evidence indicates that it is not located near the termini. Acknowledgment.-The authors wish t o acknowledge the technical assistance of Mr. A. Gross.
are accounted for by two peptides isolated from the products of chymotryptic digestion of corticotropinA and previously shown6to comprise a large section of the carboxyl end of the intact molecule. These peptides, together with pertinent data are shown in Table 11. The first peptide of Table I1 represents the last two amino acid positions in corticotropin-A and the second peptide extends from a point close to the center of the molecule out to the third position from the carboxyl end. The problem of arranging the peptides of Table I in the structure of corticotropin--4 was one of finding overlapping sequences. To this end, the long peptide of Table I1 was subjected to partial acid hydrolysis. The reaction was carried out by heating the peptide in 12 N hydrochloric acid a t 37” for 72 hours. The resulting mixture was separated by paper chromatography and among the fragments were those listed in Table 111. Fragment Yo. 1 ran very rapidly in both solvent systems and was clearly separated from all other ninhydrin-positive spots. Even without structural work this peptide (9) H. Neurath and G. W. Schwert, Chcm. Reo., 46, 69 (1950). clearly provided an overlap between fragments no. (10) F. Sauger and H. Tuppy, Biochcm. J.,49, 481 (1951). 2 and no. 3 of Table I and indicated an over-all THEARMOVR LABORATORIES arrangement of 1-3-4-2 in the fragments of cortiCHICAGO, ILLINOIS cotropin-A. Fragment no. 2 of Table I11 was more difficult to separate and identify. After the second uni-diStudies on Pituitary Adrenocorticotropin. IX. mensional chromatogram, the spot a t Leu-/0.72 showed alanine, leucine and phenylalanine after Further Sequences Near the C-Terminus complete acid hydrolysis, in the molar ratio : 2 : 1 : 1. B Y W. F. WHITE I n view of the known structures of the fragments of Table I and in view of the tentative over-all arRECEIVED APRIL17, 1954 rangement of these fragments, it was not possible to Short-term (2-4-hour) peptic hydrolyses of corti- devise a logical single sequence containing two alacotropin--~give rise to only four peptide fragments nines, one leucine and one phenylalanine. Thus the showing significant ninhydrin-positive spots in pa- Leu-/0.72 spot of Table I11 was adjudged to be a per chromatography. In these short-term hydrol- mixture of two dipeptides, each containing alanine. yses there is little, if any, loss of physiological activ- In terms of the tentative over-all arrangement, the ity as measured by the Sayers test, although pep- most likely mixture was one of Leu-Ala and *4la. sin does cause destruction of activity if the action is Phe. The former of these peptides was a t hand7 prolonged.‘ Table I lists these peptic fragments and its Rfvalues exactly fitted the data. By test together with the pertinent chromatographic, it was found that Leu.*4la was not split appreciably chemical and enzymatic data. Fragment No. 1, the by carboxypeptidase on 24-hour incubation. On slowest moving spot, is the only one from which the other hand, previous experience with a variety physiological activity has been recovered2 and con- of natural and synthetic dipeptides, indicated that tains all the amino acids found in corticotropin-A Ala.Phe would be completely split by carboxypepwith the exception of leucine. This fragment ap- tidase in 24 hours, Accordingly, the Leu-/0.72 parently is the corticotropin-B of Brink, et ~ 2 1 . ~spot of Table I11 was subjected to 24-hour treatFragment no. 2 is the tetrapeptide previously ment with carboxypeptidase. A chromatogram of shown4 to represent the last four amino acids a t the the product in the Partridge system showed alacarboxyl end of corticotropin-4. Since neither of nine, phenylalanine and a spot above phenylalanine the remaining two peptides contains serine, previ- a t Rf = 0.72 which, on complete acid hydrolysis, ously shown5 to be the N-terminal amino acid gave only alanine and leucine. Thus the second of corticotropin-& these sequences must also occur overlap (between fragments 4 and 3 of Table I) was near the carboxyl end of the intact hormone. This confirmed. conclusion is strengthened by the fact that all of the Table IV sumniarizes all of the work published amino acids of the last three peptides of Table I to date by this Laboratory on the C-terminal and of Corticotropin-il. The notation in this and in the ( I ) Under the enzymatic conditions used in this Laboratory (cf. other tables is that of Sanger.8 Table I ) , losses of physiological activity become significant at about the sixth hour and reach almost 100% at the twenty-fourth hour. Acknowledgment.-The author wishes to ac( 2 ) W. F. White, W. L. Fierce and J. V. Lesh, Proc. SOC.E x p f l . Biol. .Wed., 78, 616 (1951). (3) N. G.Brink, cf a i . , THISJOURNAL, 74, 2120 (1952). (4) W F . White, ibid., 75, 4877 (1953). ( 6 ) I\’. A . Landmann, M. P. Drake and W. F. White, i b i d . , 75, 4370 [ 1953).
(6) W. P. White end W. A. Lendmann, ibid., 78, 4193 (1954). (7) Kindly furnished by Dr. Sidney W. Fox of Iowa State College. Ames, Iowa. ( 8 ) “Advances in Protein Chemistry,” Vol. VII, Academic Press, Inc., New York, N. Y.,p. 6.
NOTES
Aug. 20, 1954
PEPTIDE FRAGMENTS SEPARATED FROM Spot R f values (peptide) s-But.no. Part.6 NHse
THE
4195
TABLE I PRODUCTS OF SHORT-TERM (2-4-Hom) PEPSIN DIGESTSOF CORTICOTROPIN-A' Order of release of amino acids by carboxypeptidased
h--Terminal determination
Amino acids on acid hydrolysis
Sequence
Phe, Val, Met, Tyr, Pro, His, Glu. Asp, DNFB:Ser None ............. Ala, Arg, Lys, Gly, Ser (also T r y by spectrophotometry) Pro.Leu.Glu,Phe Pos. isatin :Pro Phe, Glu 2 .84 Tyr Pro, Leu, Glu, Phe Phe, Ala Ala.Glu.Ala.Phe DNFB:Ala 3 .70 Arg+ Ala, Glu, Phe Asp.Glu.Leu D N F B :Asp Leu GluLeu, Asp, Glu 4 .66 a Digestion carried out a t 37" in 0.1N formic acid (pH 2.3) at corticotropin-A concentration of 5 mg./ml. with 1% pepsin crystalline A m o u r . * n-Butanol-water-acetic acid (4:s:1). s-Butyl alcohol-ammonia ( 3 :1). For details on the use of this system, cf.: J . F. Roland and A. M. Grow, Anal. Chem., in press. Since this system is used over an extended period C-Terminal with a pad a t the bottom of the sheet, RJ values are given in terms of the the nearest reference amino acid. work was done by incubating the peptide a t 37" in 0.1N ammonium acetate with approximately 1% crystalline carboxypeptidase in the presence of diisopropyl fluorophosphate. The reaction was followed a t intervals over a 20-hour period. The sequences were deduced from the order of appearance of the amino acids.
1
0.05
Two spot (peptide) no.
OF THE
Zero
PEPTIDE FRAGMEXTS SEPARATED FROM
TABLE I1 PRODUCTS OF CHYMOTRYPTIC DIGESTIONO F CORTICOTROPIN-A'
THE
R f values .4mino acids on acid hydrolysis
s-But.-
Part.
NHI
0.65 0.4-0.6
Arg+ Zero
S-Terminal determination
Order of release of amino acids by carboxypeptidase
Sequence
Glu, Phe D S F B :Glu Little, if any, splitting Glu.Phe Lys, Val, Pro*, Gly, Ala*, D S F B : L y s Leu Lys(Va1, Pro*, Gly, Ala*, 2 Asp*, Glu*, Phe).Leu Asp*, Glu*, Leu*. Phe 5 Quantitative results indicate that these amino acids are present as more than one residue. 1
TABLE 111
T\VO
O F THE PEPTIGE
FRAGMENTS SEPARATED
F R O M THE PRODUCTS O F P A R T I A L
ACID" HYDROLYSIS O F PEPrIDE
NO.
2,
TABLE I1 Spot (peptide) no.
R f values Part.
s-But.-NHa
Amino acids on acid hydrolysis
Order of release of amino acids by carboxypeptidase
1 . 4 X Phe Ala, Pro, Leu, Phe Leu 0.i2 Leu Ala, Leu
