for publication, September 21, 1954)

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1 CORTICOTROPINS (ACTH) IV. THE ACTION OF CARBOXYPEPTIDASE ON (u-corticotropin, AND THE C-TERMINAL AMINO ACID SEQUENCE BY J. IEUAN HARRIS AND CHOH HA0 LI (From the Hormone Research Laboratory, University of California, Berkeley, California) (Received for publication, September 21, 1954) In Paper I of this series (1) the isolation from sheep pituitary glands of a pure polypeptide, a-corticotropin (ACTH), was described. The results of amino acid and terminal group analyses (24) indicate that the a-corticotropin molecule is composed of a single polypeptide chain containing thirty-nine amino acid residues. Numerous chemical methods, described in Paper III (4), are available for the identification and quantitative estimation of the N-terminal groups of polypeptide chains, but as yet no generally applicable chemical procedures have been developed for a complementary investigation of the corresponding C-terminal groups. However, an enzymatic method of C-terminal group analysis based on the use of the proteinase carboxypeptidase was proposed by Lens (5), and this method has recently been extended and used with considerable success in many laboratories for the identification and estimation of C-terminal groups in polypeptide and protein molecules (6, 7). The development of a convenient micromethod (8) for the analysis of amino acids as their dinitrophenyl derivatives, which has been used to follow the rate of release of amino acids from a polypeptide chain by the action of carboxypeptidase (9, lo), has facilitated the further application of this enzymatic procedure to the elucidation of amino acid sequences from the carboxyl end. The use of carboxypeptidase for such investigations is based upon the assumption that the enzyme, by analogy with its action on synthetic substrates, attacks specifically only those bonds which are adjacent to free (r-cooh groups in polypeptide chains. It follows that the carboxypeptidase method may be applied to determine the sequence of amino acids from the carboxyl end in a protein molecule, provided that the protein substrate does not contain more than one open polypeptide chain and that the C-terminal peptide bond as well as other points of interaction with the enzyme, such as the adjacent peptide bond and the C-terminal amino acid side chain, are accessible and conform to the specificity requirements of the enzyme. With these conditions fulfilled, the enzyme would be expected to release amino acids one by one along a peptide chain, until a structural barrier is encountered as a result 499

2 500 CORTICOTROPINS. IV of failure on the part of a potentially susceptible bond to conform to the specificity requirements necessary for the continued action of the enzyme. Furthermore, as discussed by Neurath et al. (ll), since the intrinsic rate of hydrolysis of a peptide bond depends largely on the nature of the side chain of its C-terminal amino acid, the rate at which each successive amino acid is released from a polypeptide chain will be directly related to their sequence along the chain if they are arranged in order of decreasing rates of hydrolysis. At any point of inversion of this sequence the rate of hydrolysis of the preceding bond would become the rate-determining step for the hydrolysis of the succeeding one, irrespective of the nature of its side chain; hence the two adjacent amino acids should be released at equal rates. However, in the case of a peptide bond in a long chain polypeptide or protein molecule, this interpretation may not be strictly valid, since its rate of hydrolysis may also be influenced markedly by the over-all environmental conditions imposed on the bond (e.g., steric and electrostatic factors, hydrogen bonding, etc.) by virtue of the size and spatial configuration of the polypeptide chain in a folded molecule. With the above considerations in mind, the action of carboxypeptidase on ar-corticotropin has been studied in an attempt to ascertain the nature of the C-terminal residue and to determine the sequence of amino acids from the carboxyl end of the chain in the polypeptide hormone. Preliminary reports of these results have been published (3, 12). EXPERIMENTAL cu-corticotropin trichloroacetate was prepared from sheep pituitary glands as described in Paper I (1) and was found to behave as a single substance when examined by zone electrophoresis on starch, partition chromatography, and counter-current distribution. The enzyme used during the course of this work was a preparation, six times recrystallized, obtained through the courtesy of Dr. W. M. Thompson of Armour and Company. Just before use, the carboxypeptidase crystals were washed with distilled water to remove adsorbed amino acids and brought into solution at ph 8.0 to 8.5 by the dropwise addition with stirring of 1 per cent NaHC03 at O-4. A 50-fold excess of diisopropyl fluorophosphate (DFP) was added to destroy the last traces of endopeptidase activity in the carboxypeptidase preparation (13-15). Qualitative XtudiesWhen a 1 per cent solution of a-corticotropin (0.2 pmole) was allowed to react with carboxypeptidase (enzyme-substrate mole ratio, 1: 100) at ph 8.5 and at a temperature of 25 for 30 minutes, only one amino acid, phenylalanine, could be detected when the whole digest was applied to paper and submitted to chromatography in butanol-acetic acid-hz0 (4: 1:5). When the period of digestion was lengthened to 16

3 J. I. HARRIS AND C. H. LI 501 hours, however, smaller quantities of two other amino acids, glutamic acid and leucine, also were identified in a similar manner. These amino acids were released much more slowly than the phenylalanine, and on the basis of the assumption that a-corticotropin is composed of only one peptide chain it was tentatively inferred that phenylalanine occurs as its C-terminal residue and that glutamic acid and leucine occupy adjacent positions in the chain in an undetermined sequence. Semiquantitative Estimation of Liberated Amino Acids--In order to obtain a rapid semiquantitative estimate of the amino acids released by the action of carboxypeptidase on a-corticotropin, the molecular sieve procedure of Partridge and Thompson (16) was used. a-corticotropin trichloroacetate (0.2 per cent solution, 0.2 pmole) was incubated with carboxypeptidase (enzyme-substrate mole ratio, 1:SO) at 40 for 16 hours at ph 8.5. The addition of 50 mg. of Amberlite IR-120 resin (H form, 20 to 50 mesh, 8 per cent cross-linked) served to terminate the enzyme reaction by lowering the ph of the reaction solution to 3. The suspension containing the resin was shaken mechanically for 1 hour and the supernatant solution removed by means of a pipette; the resin was washed free of residual peptide with distilled water and then with 5 M ammonia (0.5 ml.) to displace the adsorbed amino acids. The ammonia eluate was taken to dryness in vacua over concentrated HzS04, redissolved in distilled water (20 pl.), and examined by two-dimensional chromatography on paper in butanol-acetic acid-ha0 (4: 1:5) and 1: 1 phenol-m-cresol saturated with borate buffer at ph 9.3 (17). As shown in Fig. 1, phenylalanine, glutamic acid, and leucine are present in approximately equimolar proportions, and presumably arise through the stepwise degradation of the cr-corticotropin molecule from the COOH end. The yield of the C-terminal amino acid phenylalanine was estimated to be approximately 0.15 to 0.20 pmole per 0.2 pmole of corticotropin by comparing its ninhydrin color with that of a graded scale of reference spots of phenylalanine (cf. Gladner and Neurath (18)). Quantitative C-Terminal Group and Sequence Analysis-In order to obtain a quantitative determination of the three amino acids which are released by the action of carboxypeptidase and to ascertain the sequence in which they occur, an additional experiment was designed to follow the rates of their release during the enzymatic reaction. a-corticotropin trichloroacetate (0.56 mg. of N) was incubated with carboxypeptidase (0.04 mg. of N; enzyme-substrate mole ratio, 1: loo), in a total solution of 5.0 ml. at ph 8.5 and 40 ; a constant ph of 8.5 was maintained throughout the course of the enzymatic reaction by the periodic addition of small amounts of 0.05 N KOH from a micro burette. Aliquots of the reaction solution (1.0 ml. containing mg. of Ly-corticotropin N) were removed after 0.5, 2, 4, 8, and 18 hours and acidified

4 502 CORTICOTROPINS. IV to ph 3.0 with 0.1 N HCl to terminate the action of the enzyme. Each aliquot was subsequently allowed to react with 0.1 ml. of dinitrofluorobenzene (FDNB) at ph 9.0 and 40 with vigorous stirring in order to maintain saturation with respect to the reagent (8); these reactions at constant ph were carried out in the reaction cell of a Jacobsen-Leonis autotitrator (19). After the reaction had proceeded for 90 minutes, the contents of the reaction vessel were transferred quantitatively to a centrifuge tube (15 ml.), and excess FDNB was extracted by shaking with three 5 ml. portions of peroxide-free ether. The aqueous layer was then -/ I Im CRESOL/PHENOL. ph 93 FIG. 1. Amino acids released from or-corticotropin by the action of carboxypeptidase. adjusted to ph 1 with 1 N HCI, and the 2,4-dinitrophenyl (DNP) derivatives of those amino acids liberated during the carboxypeptidase reaction were separated from the DNP derivative of residual a-corticotropin by means of thorough extraction of the acidified suspension with 5 ml. portions of peroxide-free ether. The DNP peptide was collected by centrifugation, and the combined ether extracts were taken to dryness in VQCUO. Dinitrophenol (formed by hydrolysis of FDNB at ph 9.0) was removed by sublimation in vucuo at 60 onto a cold finger containing solid CO2 (20). The residual DNP amino acids were analyzed by two-dimensional chromatography on paper in the toluene -1.6 M phosphate buffer system (8). A typical chromatogram (derived from the 18 hour aliquot) is shown in Fig. 2; as expected, spots were obtained corresponding to the DNP derivatives of phenylalanine, glutamic acid, and leucine. Each DNP amino acid was PHE

5 J. I. HARRIS AND C. H. LI 503 eluted separately from each chromatogram with 1 per cent NaHC03 (4 ml.), and its quantity estimated from the ultraviolet absorption at 360 rnp. In order to ascertain the extent of recovery in the presence of DNP-orcorticotropin of phenylalanine, glutamic acid, and leucine as their DNP derivatives, a control experiment was designed to simulate the conditions employed during the procedure used for the release and subsequent estimation of the amino acids formed during the reaction between a-corticotropin Toluene P GIU FIG. 2. Amino acids released from a-corticotropin by the action of carboxypeptidase, identified as their DNP derivatives. DNP OH Phe and carboxypeptidase. cr-corticotropin trichloroacetate (0.5 mg. of N) was dissolved in water (5 ml.) containing 1 pmole each of phenylalanine, glutamic acid, and leucine. The solution was incubated at ph 8.5 and 40 for 90 minutes. After removal of excess reagent, the solution was acidified to ph 1.0 and the DNP amino acids were recovered by extraction; triplicate aliquots of the ether extract (corresponding to one-tenth of the total extract) were submitted to two-dimensional chromatography on paper, eluted with 1 per cent NaHC03, and their optical densities determined at 360 rnp as described above. The results obtained, together with the calculated effective millimolar extinction coefficients (8) for the three DNP amino acids, are recorded in Table I. It is considered that the experi-

6 504 CORTICOTROPINS. IV mental losses (inter alia, losses due to incomplete extraction of the DNP amino acids in the presence of DNP peptide, losses on paper during chromatography, etc.) are reflected in the calculated effective molecular TABLE Effective Millimolar Extinction Coeficients of DNP-Phenylatanine, DNP-Glutamic Acid, and DNP-Leucine Prepared in Presence of cu-corticotropin and Subsequently Chromatographed on Paper I DNP amino acid Phenylalanine. Glutamic acid.. Leucine. Optical density at 360 m Sample It 1 Sample Zt / Sample 3t 1 Mean I * As defined by Levy (8). t 0.1 rmole aliquot. $ The values in parentheses are standard values taken from Levy (8). A LEUCINE Effective nm extinction coefficient* 14.2 (15.2)$ 16.6 (16.6) 14.4 (14.2) i lb I4 I6 I8 HOURS FIG. 3. Rate of release of amino acids from cu-corticotropin by the action of carboxypeptidase, estimated as their DNP derivatives. extinction coefficients; consequently, these latter values have been used for calculating the yields, in micromoles of the amino acids liberated in the carboxypeptidase reaction, from corresponding optical density readings. Results showing the rates of release of phenylalanine, glutamic acid, and leucine from a-corticotropin are presented in Fig. 3. The C-terminal amino acid, phenylalanine, is completely released under the conditions of

7 J. I. HARRIS AND C. H. LI 505 the experiment and attains a plateau value corresponding to pmole (~to.006) from 0.66 mg. of ol-corticotropin. If it is assumed that 1 mole of phenylalanine is released per mole of ac-corticotropin, a value of 4300 (~t200) may be calculated for its equivalent weight; this value is in good agreement with the minimal molecular weight of cr-corticotropin (4541) calculated on the basis of its amino acid composition (2). Glutamic acid and leucine are released successively in this sequence but at much slower rates, and attain values equivalent to approximately 75 and 65 per cent, respectively, of the phenylalanine only after 18 hours of digestion. However, when cy-corticotropin which had been digested for 18 hours under the conditions described above was precipitated at ph 5.5 to 6.0 and again treated with fresh enzyme, additional amounts of glutamic acid and leucine, estimated as 0.2 to 0.3 mole of each amino acid by spot dilution chromatography (cf. (US)), were released, whereas phenylalanine, according to expectation, was now completely absent. No other amino acids were released, even after prolonged digestion with high concentrations of enzyme, an observation which would seem to indicate a structural barrier to further degradation along the peptide chain. DISCUSSION From the results of the present investigation it is concluded that phenylalanine, glutamic acid, and leucine are released successively in equivalent stoichiometric proportions when a-corticotropin is allowed to react with carboxypeptidase, and that they arise through the stepwise degradation of the C-terminal sequence...leu.glu.phe in the peptide hormone. The nature of the enzymatic reaction is consistent with what is known about the specificity characteristics of carboxypeptidase. As expected, the C-terminal phenylalanine is released rapidly despite the presence of a +y-cooh group in the side chain of the adjacent residue, glutamic acid. After the release of phenylalanine, however, glutamic acid is exposed as the new C-terminal residue; a charged group in the side chain of a C-terminal residue is known to retard the action of the enzyme (24, 25), and consequently the slow rate of release of glutamic acid was expected. In addition, the rate of release of glutamic acid should determine the rate at which the peptide bond involving the COOH group of the next amino acid along 1 a-corticotropin from which the three C-terminal amino acids (Phe, Glu, and Leu) had been quantitatively removed by the action of carboxypeptidase (after two cycles of reaction, as described above) was found to retain hormonal activity. It is significant that insulin (21), lysozyme (J. I. Harris, unpublished data), tobacco mosaic virus (22), growth hormone (IO), and cu-chymotrypsin (23) have also been found to retain their respective biological functions after the liberation of their respective C-terminal amino acid residues.

8 506 CORTICOTROPINS. IV the chain, leucine, becomes available to the enzyme. On the basis of side chain affinity, leucine with its fatty side chain should be released as rapidly as it becomes available to the enzyme, theoretically at a rate equal to that of the preceding residue, glutamic acid. However, leucine is released at a rate which is significantly slower than that of glutamic acid. The fact that no further reaction takes place after stoichiometric amounts of phenylalanine, glutamic acid, and leucine have been released in the course of the reaction with carboxypeptidase suggests that the fourth amino acid along the chain is one which does not conform to the specificity requirements of the enzyme and consequently resists digestion. Studies on synthetic substrates have shown that peptide bonds involving C-terminal proline are for all practical purposes completely resistant to the action of carboxypeptidase (25). Furthermore, in order to exert its maximal effect, carboxypeptidase requires the presence of a normal -CO-NHpeptide bond in a position adjacent to the susceptible C-terminal linkage. The presence, for example, of an adjacent peptide bond involving the ring nitrogen of proline \/ t -CO-N- 1 is known to retard, and in some cases to inhibit completely, the release of the C-terminal amino acid (24, 25). In the light of these findings, the occurrence of the C-terminal tetrapeptide sequence.pro.leu.glu.phe in the oc-corticotropin molecule could account for the apparent structural barrier to further enzymatic digestion after the quantitative liberation of the C-terminal amino acids (phenylalanine, glutamic acid, and leucine) and for the unusually slow release of leucine when it in turn becomes the C-terminal amino acid after the removal of phenylalanine and glutamic acid. Evidence which indicates that the above C-terminal tetrapeptide sequence does in fact occur in or-corticotropin was provided by the isolation and characterization from a pepsin digest of the hormone, the tetrapeptide Pro.Leu.Glu.Phe (3). The RF value of this fragment was 0.83 when it was submitted to chromatography on Whatman No. 1 paper in butanolacetic acid-hzo, 4: 1: 5 (see White (26)). Since it is known that only 1 residue of leucine is present in the molecule (2), the tetrapeptide Pro.Leu. Glu.Phe is shown to be the C-terminal sequence in a-corticotropin. It is worthy of note that corticotropin A, isolated from hog pituitary glands (27), has been reported to have the same C-terminal tetrapeptide sequence (26). SUMMARY When a-corticotropin is allowed to react with carboxypeptidase, phenylalanine, glutamic acid, and leucine are shown to be released successively and in equivalent stoichiometric amounts. From theoretical considerations of the kinetic data and the isolation of the tetrapeptide Pro.Leu.

9 J. I. HARRIS AND C. H. LI 507 Glu.Phe from a pepsin digest, it is concluded that the amino acids are released by the stepwise degradation of the C-terminal tetrapeptide sequence..pro.leu.glu.phe in the peptide hormone. The authors wish to acknowledge the able assistance of Ning G. Pon during the course of this investigation. It is a pleasure to record our appreciation of research grants from the National Institutes of Health, United States Public Health Service (No. G-2907), and from Eli Lilly and Com- pany. BIBLIOGRAPHY 1. Li, C. H., Geschwind, I. I., Dixon, J. S., Levy, A. L., and Harris, J. I., J. Biol. Chem., 213, 171 (1955). 2. Levy, A. L., Geschwind, I. I., and Li, C. H., J. Biol. Chem., 213, 187 (1955). 3. Harris, J. I., and Li, C. H., J. Am. Chem. Sot., 76, 3607 (1954). 4. Levy, A. L., and Li, C. H., J. Biol. Chem., 213, 487 (1955). 5. Lens, J., Biochim. et biophys. acta, 3, 367 (1949). 6. Jutisz, M., Bull. Sot. chim. biol., 36, 109 (1954).. 7. Harris, J. I., in Glick, D., Methods of biochemical analysis, New York, 2, in press. 8. Levy, A. L., Nature, 174, 126 (1954). 9. Anfinsen, C. B., Redfield, R. R., Choate, W. L., Page, J., and Carroll, W. R., J. Biol. Chem., 207, 201 (1954). 10. Harris, J. I., Li, C. H., Condliffe, P. G., and Pon, N. G., J. Biol. Chem., 209, 133 (1954). 11. Neurath, H., Gladner, J. A., and Davie, E. W., in McElroy, W. D., and Glass, B., The mechanism of enzyme action, Baltimore, 50 (1954). 12. Li, C. H., Geschwind, I. I., Levy, A. L., Harris, J. I., Dixon, J. S., Pon, N. G., and Porath, J. O., Nature, 173, 251 (1954). 13. Jansen, E. F., Nutting, M.-D. F., Jang, R., and Balls, A. K., J. Biol. Chem., 179, 189 (1949). 14. Gladner, J. A., and Neurath, H., Biochim. et biophys. acta, 9, 335 (1952). 15. Sanger, F., and Thompson, E. 0. P., Biochem. J., 63, 366 (1953). 16. Partridge, S. M., Nature, 169, 493 (1952). Thompson, A. R., Nature, 169, 495 (1952). 17. Levy, A. L., and Chung, D., Anal. Chem., 26, 396 (1953). 18. Gladner, J. A., and Neurath, H., J. Biol. Chem., 206, 345 (1953). 19. Jacobsen, C. F., and Leonis, J., Compt.-rend. truv. Lab. Car&berg, Skrie chim., 27, 333 (1951). 20. Mills, G. I,., Biochem. J., 60, 707 ( ). 21. Harris, J. I., and Li, C. H., J. Am. Chem. Sot., 74, 2945 (1952). 22. Harris, J. I., and Knight, C. A., Nature, 170, 613 (1952). 23. Gladner, J. A., and Neurath, H., J. Biol. Chem., 206,911 (1954). 24. Neurath, H., and Schwert, G. W., Chem. Rev., 46, 69 (1950). 25. Smith, E. L., in Wolstenholme, G. E. W., and Cameron, M. P., Chemical structure of proteins, Ciba Foundation symposium, Boston, 109 (1954). 26. White, W. F., J. Am. Chem. Sot., 76, 4877 (1953). 27. White, W. F., J. Am. Chem. Sot., 76, 503 (1953).

10 CORTICOTROPINS (ACTH): IV. THE ACTION OF CARBOXYPEPTIDASE ON α-corticotropin, AND THE C-TERMINAL AMINO ACID SEQUENCE J. Ieuan Harris and Choh Hao Li J. Biol. Chem. 1955, 213: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at tml#ref-list-1