(high-sensitivity automated Edman degradation/radio-iodination/peptide ... H. D. NIALL*, R. T. SAUER*, J. W. JACOBS*, H. T. KEUTMANN*, G. V. SEGRE*, ...
Proc. Nat. Acad. Sci. USA Vol. 71, No. 2, pp. 384-388, February 1974
The Amino-Acid Sequence of the Amino-Terminal 37 Residues of Human Parathyroid Hormone (high-sensitivity automated Edman degradation/radio-iodination/peptide synthesis/immunoreactivity)
H. D. NIALL*, R. T. SAUER*, J. W. JACOBS*, H. T. KEUTMANN*, G. V. SEGRE*, J. L. H. O'RIORDANt, G. D. AURBACHT, AND J. T. POTTS, JR.* * Endocrine Unit, Massachusetts General Hospital, Boston, Mass. 02114; t The Middlesex Hospital, London, W.1, England; and
1 Section on Mineral Metabolism, National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014
Communicated by Paul C. Zamecnik, October 9, 1973 ABSTRACT The sequence of the amino-terminal 37 residues of human parathyroid hormone has been established. The hormone used in these studies was isolated in highly purified form from parathyroid adenomata and was subjected to automated degradation in a Beckman sequencer. A high-sensitivity sequencing procedure employing 36S-labeled phenylisothiocyanate of high specific activity as the coupling agent was used. The sequence obtained differs from that of bovine parathyroid hormone in three of the first 37 positions, and from that of porcine parathyroid hormone in two positions. A single human-specific residue was found (asparagine 16). The sequence obtained differs at three positions (22,28, and 30) from the structure for human parathyroid hormone reported recently by Brewer et al. 1(1972) Proc. Nat. Acad. Sci. USA 69, 3585-35881 and synthesized by Andreatta et al. [(1973) Helv. Chim. Acta, 56, 470-4731 We have carefully reviewed our data, reported here in detail, on the sequence positions in dispute. We must conclude, on the basis of all available data, that the structure that we propose is the correct structure. The objective resolution of these discrepancies in structural analysis through further chemical and immunochemical studies is important, since synthesis of human parathyroid hormone, in which there is widespread interest for physiological and clinical studies, must be based on the correct sequence of the human hormone if the peptide is to be genuinely useful.
Substantial advances have been made in recent years in our knowledge of parathyroid hormones, through studies of primary structure (1-3), structural requirements for biological activity (4, 5), biosynthesis (6-8), and metabolism (9-14). Most of these studies, including the development and application of radioimmunoassays capable of measuring plasma parathyroid hormone levels in man, have depended directly or indirectly upon the use of the bovine and porcine hormones. Purified human parathyroid hormone (HPTH), on the other hand, has been available in microgram quantities, sufficient only for limited studies of its chemical and immonological properties (15). Recent improvements in extraction and isolation techniques, and the development of high-sensitivity methods for peptide sequence analysis have permitted us to determine the amino-acid sequence of the amino-terminal biologically active portion of HPTH (Fig. 1). After the submission for publication in abstract form of our findings for the N-terminal 31 residues of HPTH (24), the report of Brewer et al. (29) of their own independent struc-
Abbreviations: HPTH, human parathyroid hormone; PTH, phenylthiohydantoin; TLC, thin-layer chromatography; MIH, mono-iodohistidine; DIH, di-iodohistidine. 384
tural studies on HPTH was published. Marked discrepancies between the two structures, which differ in three of the first 30 residues, have prompted us to reexamine our data for each cycle of the several degradations performed with the phenylisothiocyanate method. We now report in full the strategy and methods used in our sequence analysis as well as the quantitative aspects of the results and discuss the nature, implications, and possible approaches to resolution of the differences between the findings of Brewer et al. (29) and ourselves concerning the sequence of the amino-terminal portion of human parathyroid hormone. MATERIALS AND METHODS
The HPTH used in these studies was extracted from 500 g of pooled human adenoma tissue by use of 88% phenol, followed by treatment with 6% NaCl and precipitation with trichloroacetic acid (15, 16). The hormone was further purified by gel filtration on Bio-Gel P-100 (Bio-Rad Laboratories, Richmond, Calif.) and ion-exchange chromatography on carboxymethyl-cellulose (Whatman CM-52; Reeve Angel Co., Summit, N.J.) (16). Hormone purification was monitored by radioimmunoassay (11). Automated Edman degradations were performed on the Beckman model 890 sequencer (Beckman Instruments, Palo Alto, Calif.) using the single-coupling, double-cleavage method of Edman and Begg (17), and other procedures recently described (18). Manual Edman degradations were performed as previously described (19). Reagents and solvents were obtained from Beckman Instruments. 35S-Labeled phenylisothiocyanate was obtained from Amersham/Searle (Arlington Heights, Ill.). The phenylthiohydantoin (PTH) derivatives were identified by thin-layer chromatography (TLC) on silica gel plates (Analtech, Inc., Newark, Del.) (17, 20) and by gas-liquid chromatography (21) using a two-column system (10% DC560 and 1.5% AN-600). PTH-histidine was identified by the Pauly reaction (22) and PTH-arginine by the phenanthrenequinone reaction (23). Quantitative yields of the PTH-aminoacid derivatives at each cycle of degradation were determined by comparison with known standards on gas-liquid chromatography. The [35S]PTH-amino acids were separated by TLC; the radioactive spots were identified by autoradiography and quantitated in a Packard model 3375 liquid scintillation counter (Packard Instrument Co., Downers Grove, Ill.). Mono-iodohistidine (MIH) and di-iodohistidine (DIH) were synthesized by the method of Brunnings (25) using the modifications of Savoie et al. (26). The phenylthiohydantoin
3 4 5 6
7 8 9 10 11 12 13 14 15 16 17 18 19 20
Glut Thr Trp Leu Arg
23 24 25 26
Lys
35.0 97.5 51.5 42.3
27 28 29
Lys Leu Glu*
30.6
30
Gln Asp
20.1 97.5 47.5 62.1
31 32 33
Ser Glu Ile Glu* Gln Leu Met His
85.3 71.4 108.6
Asp* Asn Leu Gly Lys His Leu Asp* Asn Ser Met Glu Arg
27.6 55.5 17.7 18.1 43.0 34.8 20.2 16.4
20.7
34 35 36
37 38 39 40
Ala
1.2
SER
L
0L
LY
L
*T
22
t
SER
bOLY
%%t
A
VAL %6LU 8' 0
HIS
O 20 F
MET 0
%
LEU
*%' 6LU
E c
*
10 9oic
0 U* LYSLts * AR LO
-~0 .11%.
w
5:-
FIG. 2. Yields of phenylthiohydantoinamino acids obtained during automated degradation of native human parathyroid hormone. See Table 1 and text.
VAL 0
0
GLNI
5
.
I
RE
ASP
.
.00 0 AN
VAL
4 E
1%
ALA 0
4
23
E
*%ASN
50FO' HIS GLN
0
10 11 12 13 14 15 16
~LEU
*
SER0
9
RESULTS Purified HPTH (140 nmol) was subjected to automated Edman degradation for 40 cycles. The PTH-amino acid derivatives identified at each cycle of this degradation, and their yields, are presented in Table 1. To illustrate repetitive yield these results are also plotted in Fig. 2. As shown in Table 1, unique amino-acid assignments, and quantitation of the single residue identified, were possible at all but two of the first 37 cycles of this degradation. At cycle 22, evidence was obtained for two residues, threonine and glutamic acid. Since the quantitative recovery of both of these residues can be low, further experiments were performed prior to definitive assignment of position 22. At cycle 32, a rise in PTH-serine above background levels was observed. However, its yield (Table 1) was considerably below that expected, even for the labile phenylthiohydantoin derivative of serine (18). Although histidine is present at this position in porcine and bovine parathyroid hormones, this residue could not be detected either by the Pauly method or by a definite increase in radioactivity associated with [85SJPTH-histidine at this cycle. However, since the overall yield at this stage of the degradation was near the detection limits for histidine by these methods, further experiments were performed prior to assignnent of this position. The presence of methionine at positions 8 and 18 of the native hormone accounted for both methionines found by amino-
derivatives of MIH and DIH were prepared as described by Edman (27). PTH-MIH and PTH-DIH were separated from all other PTH-amino-acid derivatives by TLC in the solvent system n-butyl acetate: water: propionic acid: formamide (240:200:30: 60). PTH- [12'I]MIH and PTH-[1251]DIH were identified by cochromatography with their respective 127I derivatives followed by autoradiography, and quantitated by counting in a Packard model 3001 gamma well spectrometer. ILE
8
Cleavage of the hormone with cyanogen bromide (CNBr) was carried out in 70% formic acid for 12 hr, 200, with a 100-fold molar excess of CNBr. Digestion with TPCK-trypsin (Worthington Biochemical Corp., Freehold, N.J.) was performed in 0.2 M trimethylamine acetate buffer (pH 9.2) at 370, for a period of 2 hr using an enzyme-to-substrate ratio of 1/100.
* Partial deamidation during the conversion reaction accounts for the presence of the free acid as well as the amide form at positions 6 and 29 (glutamines) and positions 10, 16, and 33 (asparagines). t See text for discussion. t TLC identification.
I O O _s
7
FIG. 1. The amino-terminal 37 residues of human parathyroid hormone.
9.1 0.7 2.7
2.1
6
3T 36 35 34 33 32 31 30 29 28 27 26 25 24
15.9 11.0 3.1 2.8 4.5
Leu
5
... LEU-ALA-VAL-PHE-ASN-HIS-VAL-ASP-GLN-LEU-LYS-LYS-ARG-LEU-TRP-GLU
18.7 13.0 14.6
3.0 3.0 1.4
4
SER 17 MET 18 GLU 19 ARG 20 VAL 21
10.5 7.3 10.1
Val Sert Asp* Asn Phe Val Ala
3
NH2-SER-VAL-SER-GLU-ILE-GLN-LEU-MET-HIS-ASN-LEU-GLY-LYS-HIS-LEU-ASN
PTH-amino acids Yield (nmol) Found Cycle 23.8 Val 21 22
2
1
TABLE 1. Automated Edman degradation of native HPTH PTH-amino acids Yield (nmol) Cycle Found 80.0 Ser 1 130.2 Val 2
385
Human Parathyroid Hormone
Proc. Nat. Acad. Sci. USA 71 (1974)
8
12
16
24 20 RESIDUE
28
32
36
40
386
Biochemistry: Niall et al.
Proc. Nat. Acad. Sci. USA 71 (1974)
TABLE 2. I')
0
0
0.
0.
Cycle CNBrl
0L
1 2 3
Serl Val Ser
FIG. 3. Release of radioactive (125I-labeled) phenylthiohydantoin derivatives of mono- and di-iodohistidine during automated degradation of 125I-labeled human parathyroid hormone. The break in the horizontal axis is introduced to simplify the presentation. No significant release of histidine-associated radioactivity was seen in cycles 17-28. [l26I]Histidine was found at cycles 9, 14, and 32, indicating the presence of histidine at these positions. See text. Numbers on the ordinates are to be multiplied by the indicated factors to obtain the experimental values.
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Glu Ile Gln Leu Met§ His Asn Leu Gly -
c)
7
8
9 10
12 13
11
4 15
16
29 30
31
32 33 34
NUMBER
CYCLE
acid analysis (16). This indicated that cleavage of the human hormone with CNBr should result in generation of three principal peptides representing residues 1-8, 9-18, and 19carboxyl terminus of the native hormone. HPTH (27 nmol) was cleaved with CNBr and the unfractionated peptide mixture subjected to 19 cycles of Edman degradation. The results are presented in Table 2. The expected three end-groups, Ser1, His9, and Glu'9 were identified at cycle one of the degradation. At cycle 4 of the degradation, corresponding to residues 4, 12, and 22 of the intact hormone, only PTH-Glu and PTH-Gly were observed in significant yield. No threonine was detected at this cycle. Therefore, glutamic acid was assigned as residue 22 of native HPTH. The significance of the finding of threonine in the amino-terminal degradation remains uncertain. As can be seen in Table 2, the results of the CNBr mixture analysis also provided complete confirmation of all r6sidue assignments made on the basis of the aminoterminal degradation on intact HPTH. Since the limited supply of purified HPTH excluded the use of conventional protein chemical methods for reexamination of position 32, an alternative radioactive micro-method was developed to permit detection of histidine residues. Purified HPTH (0.75 ,ug) was iodinated with IuI by a modification of the Hunter-Greenwood procedure (28). Unlabeled bovine parathyroid hormone was then added as carrier and the mixture was degraded in the sequencer. At each cycle, the radioactivity migrating with PTH- [127I ]MIH and PTH- [1'7I ]DIH on TLC was determined. These data (Fig. 3) demonstrate
*
Glu Trp Leu Arg Lys Lys Leu
Yield of PTH Derivative (nmol) Ser 18.3, His*, Gly 27.0 Val 11.7, Asn 4.3, Argt Ser 10.2, Leu 8.5, Val 18.4 Glu 4.5, Gly 3.1 Be 10.2, Lys$, Trp 58 Gln 4.9, His*, Leu 10.5 Leu 9.8, Argt Met 3.4, Asnt, Lyst His*, Ser 4.2, Lyst Asnt, Leu 7. 5
Gln
Leul.4,Gln4.6
Asp Val
Gly 0.9, Asp 2.3 Val 4.2
Asn Phe Val Ala Leu
Asnt Phe 2.6 Val4.5 Ala 2.0 Leu 2.0
CNBr2
CNBr3
His9 Asn Leu
Arg Val
Gly
Lys His Leu Asn Ser
Glu19
-
-
-
Identification by Pauly reaction.
t Identification by phenanthrenequinone reaction. t Identification by thin-layer chromatography. § Presence of methionine at cycle 8 with the following four residues obtained at cycles 9-12 indicates that cleavage of the Met8His9 bond by cyanogen bromide was incomplete. Residues from this sequence, presumably representing the 1-18 peptide fragment, could not be detected subsequent to cycle 12. ¶ Histidine, subsequently found to occupy position 32 (see text) was not detected at the expected cycle (number 14) of this degradation.
the presence of histidine at cycle 32, and confirm the histidine at cycles 9 and 14. To confirm these results further, and in particular to examine the differences between Brewer et al. (29) and ourselves concerning the nature of residue 28, iodinated HPTH was digested with trypsin and then subjected to Edman degradation for seven cycles. The PTH derivatives of [125I]MIH and 1251I]DIH were found at cycles 1 and 5 of the degradation Fig. 4). The histidine at cycle 1 further confirmed the Lys"3-His" sequence already determined. The finding of histidine at cycle 5 would be predicted on the basis of tryptic cleavage carboxyl to Lys27, and therefore both supports the assignment of His32 and argues against the report of Brewer et al. (29) that residue 28 is lysine. DISCUSSION
to)
9-
0
10.
(0
3.
0
1
2
s 6 7 NUMBER
3 4
CYCLE
FIG. 4. Release of radioactive (12I-labeled) phenylthiohydantoin derivatives of mono- and di-iodohistidine during degradation of a tryptic digest of iodinated HPTH. [125I]Histidine was released at cycles 1 and 5. See text.
The amino-terminal sequence we propose for HPTH differs from that of both the bovine and the porcine hormones. HPTH differs from bovine parathyroid hormone (Fig. 5) at positions 1, 7, and 16. The porcine and human hormones differ at positions 16 and 18. Asn16 is the only unique residue found in the active region of human parathyroid hormone. Unexpectedly, our structure differs at three positions from that recently proposed by Brewer et al. (29) for the aminoterminal 34 residues of HPTH. They report residue 22 to be glutamine, residue 28 to be lysine, and residue 30 to be leucine. If correct, all these changed residues would be unique to the human hormone. In contrast, we find residue 22 to be glutamic
Proc. Nat. Acad. Sci. USA 71
MT
~-~3.!.
Human
(1974) HI~
.Y.5 LEJ ASNI
Parathyroid Hormone
387
0.
0
0
I cI
I-
-l In
o
PORCINE.t
X '< aim:
I, 35
/
I
a.
28 29 30 31 32 26 27 28 29 30 CYCLE NUMBER CYC L E NUMBER FIG. 6. Yields of PTH-leucine at cycles 26-30 and of PTHaspartic acid at cycles 28-32 obtained during automated degradation of native human parathyroid hormone. A sharp rise above background levels is seen at cycle 28 for leucine and at cycle 30 for aspartic acid. See text.
* ** HUMAN BOVINE
i
FIG. 5. Comparison of the amino-terminal sequences of porcine, bovine, and human parathyroid hormones. The central continuous sequence is that of the human hormone (residues 1, 7, and 16). Residues differing in the bovine hormone are stippled; those differing in porcine parathyroid hormone (16 and 18) are hatched.
acid, residue 28 to be leucine, and residue 30 to be aspartic acid. The residues we have identified are identical with those at the corresponding positions in both the porcine and bovine hormones. Since both groups have isolated the hormone from essentially similar sources, i.e., human adenoma tissue pooled from many centers, the possibility that there are two hormonal forms which differ as markedly as those of the two proposed sequences is highly unlikely. We have carefully reexamined our data from both degradations with particular emphasis on the positions in question. In neither degradation was any glutamine observed at cycles corresponding to residue 22. Glutamine and asparagine can undergo deamidation during degradation. However, in both degradations (Tables 1 and 2) glutamine and asparagine residues were detected at cycles beyond position 22, making it implausible that the glutamic acid detected at position 22 was originally glutamine. At cycles corresponding to residues 28 and 30, leucine and aspartic acid were clearly identified by their predominant yields (Tables 1 and 2). The relative rise in yield of these residues above and subsequent fall to background levels is shown in Fig. 6. In automated Edman degradation the phenomenon of increasing overlap, which tends to be cumulative from cycle to cycle, has been well documented (17, 18, 30). Edman and Begg have, however, found that use of a double-cleavage program can limit this overlap to relatively low levels (17) even in extended degradations. In our amino-terminal degradation, which employed such a double-cleavage program, overlap rose from 4.5% at cycle 12 to 14.5% at cycle 28. Brewer et al. reported quantitative data only at position 12; their data permit the calculation that there was a 32% overlap at this early phase of degradation. The natural increase of this already substantial overlap, particularly in view of their use of a single-cleavage program, would make assignment of repeating residues at later cycles of the degradation, such as the putative Lys28 in a sequence Lys26-Lys27-Lys28, particularly hazardous. If, as proposed by Brewer et al. (29), residue 28 is lysine, tryptic digestion of [1251]HPTH would lead to cleavage of the
hormone carboxyl-terminal to residue 28. Therefore, the PTH derivatives of [1J5I]MIH and [125I]DIH corresponding to His32 would be released at the fourth cycle of degradation, rather than the fifth. Clearly, however, histidine is released only at the first and fifth cycles (Fig. 4). Ultimate resolution of the differences in the proposed structures must await further studies. However, a comparison of our results and methods with the published results and methods of Brewer et al. (29) leads us to conclude that our proposed structure based on a variety of approaches is more likely to be correct. Tests of the biological and immunological properties of synthetic peptides corresponding to our structure and to the structure proposed by Brewer et al. (29) for the amino-terminal portion of human parathyroid hormone may prove helpful in objectively resolving the discrepancies in the structures proposed. The marked differences, which include a change in net charge of three within a sequence of nine residues, might affect biological activity. Even more likely is the possibility that such charge differences will result in clear-cut differences in immunoreactivity when the synthetic peptides based on the two proposed structures are each compared with native human parathyroid hormone in their ability to combine with antisera directed against the amino-terminal region (11). If the structure of Brewer and associates is, as we believe, incorrect, use of antisera generated against the corresponding synthetic peptide for radioimmunoassay studies could confuse rather than aid attempts to more accurately measure HPTH or to understand the complex pattern of metabolism of parathyroid hormone (11). A preliminary immunoassay study based on the peptide of Brewer et al. (29) and Andreatta et al. (34) has already been published (35). Clearly it is extremely important to establish whether our sequence or that of Brewer et al. (29) represents the native HPTH structure before various laboratories embark on extensive immunological studies using synthetic HPTH peptide. An amino-terminal tetratriacontapeptide based on the structure proposed here has been synthesized by the solid-phase method (31). Studies of the potency of this peptide as measured in vitro by activation of renal-cortical adenylate cyclase indicate that its activity is 1030 units/mg, closely equivalent, on a molar basis, to the potency of 350 units/mg (16) for native human parathyroid hormone in this assay. Assays in vivo using the chick hypercalcemia assay (32) indicate a potency of 7000 units/mg, an activity identical to that of the bovine peptide 1-34 (no native human parathyroid hormone was available for assay in this system).
388
Biochemistry: Niall et al.
The immunological activity of the synthetic peptide has been examined with several antisera directed against the amino-terminal region of parathyroid hormone. Tests against antiserum 199 (33) and GP-1 (11) indicated that reactivity on a molar basis of our synthetic peptide was identical qualitatively and quantitatively to that of the native human hormone. No details have been reported on the specific biological or immunological activity of the synthetic peptide of Andreatta et al. (34), whose structure was based on the structure reported by Brewer et al. (29). Comparisons based on detailed biological and immunological tests of the two synthetic peptides in various laboratories should be of considerable interest. Our present findings carry several implications for the structural and comparative immunochemical studies of human parathyroid hormone. The sequence of HPTH we find is identical with that of either the bovine or porcine hormone at 36 of the first 37 residues; the changes found do not affect net charge and do not greatly alter physicochemical properties. Hence, although some improvements in detection of human parathyroid hormone might result from use of antisera directed against the amino-terminal sequence of the human hormone, the improvements, in our view, might not be large. In fact, the success encountered already in numerous laboratories in detection of the human hormone with immunoassays based on the bovine molecule is consistent with the overall chemical similarity found in the amino-terminal sequences of the three species of parathyroid hormone. On the other hand, previous immunochemical and analytical evidence (11, 15, 16) indicates that more marked differences in structure between bovine and human hormones are likely to be found in the carboxyl-terminal region. Since a large carboxyl-terminal fragment appears to be the major form of immunoreactive parathyroid hormone in the human circulation (11), antisera that recognize the carboxyl end of the human hormone are most likely to significantly improve immunoassay sensitivity. Further sequence studies on HPTH, followed by synthesis of selected peptides from the carboxylterminal two-thirds of the molecule, may well result in antisera considerably more sensitive for detection of human parathyroid hormone. Collection of the human parathyroid adenomata used in this study was made possible through the cooperation of many individuals and institutions in the United States, Canada, and overseas. Special thanks are due the Medical Research Council of Great Britain for help with this project. This investigation was supported in part by Grants AM 11794 and AM 04501 from the National Institute of Arthritis, Metabolic and Digestive Diseases. G.V.S. is the George Morris Piersol Teaching and Research Scholar of the American College of Physicians and Special Fellow of the National Institute of Arthritis, Metabolism and Digestive Diseases. 1. Niall, H. D., Keutmann, H. T., Sauer, R., Hogan, M. L., Dawson, B. F., Aurbach, G. D. & Potts, J. T., Jr. (1970) Hoppe-Seyler's Z. Physiol. Chem. 351, 1586-1588. 2. Brewer, H. B., Jr. & Ronan, R. (1970) Proc. Nat. Acad. Sci. USA 67, 1862-1869. 3. O'Riordan, J. L. H., Woodhead, J. S., Robinson, C. J., Parsons, J. A., Keutmann, H. T., Niall, H. D. & Potts, J. T., Jr. (1971) Proc. Roy. Soc. Med. 64, 1263-1265. 4. Keutmann, H. T., Dawson, B. F., Aurbach, G. D. & Potts, J. T., Jr. (1972) Biochemistry 11, 1973-1979. 5. Potts, J. T., Jr., Tregear, G. W., Keutmann, H. T., Niall, H. D., Sauer, R., Deftos, L. J., Dawson, B. F., Hogan, M. L. & Aurbach, G. D. (1971) Proc. Nat. Acad. Sci. USA 68, 63-67.
Proc. Nat. Acad. Sci. USA 71 (1974) 6. Tregeor, G. W., van Rietschoten, J., Greene, E., Keutmann, H. T., Niall, H. D., Reit, B., Parsons, J. A. & Potts, J. T., Jr. (1973) Endocrinology 93, 1349-1353. 7. Cohn, D. V., MacGregor, R. R., Chu, L. L. H. & Hamilton, J. W. (1972) in Calcium, Parathyroid Hormone and the Calcitonins eds. Talmage, R. V. & Munson, P. L. (Excerpta Medica, Amsterdam, The Netherlands), pp. 173-182. 8. Kemper, B., Habener, J. F., Potts, J. T., Jr. & Rich, A. (1972) Proc. Nat. Acad. Sci. USA 69, 643-647. 9. Habener, J. F., Powell, D., Murray, T. M., Mayer, G. P. & Potts, J. T., Jr. (1971) Proc. Nat. Acad. Sci. USA 68, 29862991. 10. Potts, J. T., Jr., Niall, H. D., Tregear, G. W., Van Rietschoten, J., Habener, J. F., Segre, G. V. & Keutmann, H. T. (1973) Mt. Sinai J. Med. 40, 448-461. 11. Segre, G. V., Habener, J. F., Powell, D., Tregear, G. W. & Potts, J. T., Jr. (1972) J. Clin. Invest. 51, 3163-3172. 12. Canterbury, J. M. & Reiss, E. (1972) Proc. Soc. Exp. Biol. Med. 140, 1393-1398. 13. Canterbury, J. M., Levey, G. S. & Reiss, E. (1973) J. Clin. Invest. 52, 524-527. 14. Goldsmith, R. S., Furszyfer, J., Johnson, W. J., Fournier, A. E., Sizemore, G. W. & Arnaud, C. D. (1973) J. Clin. Invest. 52, 173-180. 15. O'Riordan, J. L. H., Aurbach, G. D. & Potts, J. T. Jr. (1971) Endocrinology 89, 234-239. 16. Keutmann, H. T., Barling, P. M., Hendy, G. N., Segre, G. V., Niall, H. D., Aurbach, G. D., Potts, J. T., Jr. & O'Riordan, J. L. H. (1974) Biochemistry, in press. 17. Edman, P. & Begg, G. (1967) Eur. J. Biochem. 1, 80-91. 18. Niall, H. D. (1973) in Methods in Enzymology, eds. Hirs, C. H. W. & Timasheff, S. N. (Academic Press, New York), Vol. XXVII, part D, in press. 19. Niall, H. D., Keutmann, H. T., Copp, D. H. & Potts, J. T., Jr. (1969) Proc. Nat. Acad. Sci. USA 64, 771-778. 20. Morgan, F. J. & Henschen, A. (1969) Biochim. Biophys. Acta 181, 43-104. 21. Pisano, J. J. & Bronzert, T. (1969) J. Biol. Chem. 244, 55975607. 22. Easley, C. W. (1965) Biochim. Biophys. Acta 107, 386-388. 23. Yamada, S. & Itano, H. A. (1966) Biochim. Biophys. Acta 130, 538-540. 24. Jacobs, J. W., Sauer, R. T., Niall, H. D., Keutmann, H. T., O'Riordan, J. L. H., Aurbach, G. D. & Potts, J. T., Jr. (1973) Fed. Proc. 32, 2445. 25. Brunnings, K. J. (1947) J. Amer. Chem. Soc. 69, 205. 26. Savoie, J. C., Thomopoulos, P. & Savoie, F. (1973) J. Clin. Invest. 52, 106-115. 27. Edman, P. (1950) Acta Chem. Scand. 4, 277-282. 28. Hunter, W. M. & Greenwood, F. C. (1962) Nature 194, 495496. 29. Brewer, H. B., Jr., Fairwell, T., Ronan, R., Sizemore, G. W. & Arnaud, G. D. (1972) Proc. Nat. Acad. Sci. USA 69,
3585-3588. 30. Hermodson, M. A., Ericson, L. H., Titani, K., Neurath, H. & Walsh, K. A. (1972) Biochemistry 11, 4493-4502. 31. Tregear, G. W., van Reitschoten, J., Greene, E., Keutmann, H. T., Niall, H. D., Parsons, J. A. & Potts, J. T., Jr. (1974) "Principles and recent applications in the solid-phase synthesis of peptide hormones," in Endocrinology 1973: Proceedings of the Fourth International Symposium, ed. Taylor, S. (Heinemann, London), in press. 32. Parsons, J. A., Reit, B. & Robinson, C. J. (1973) Endocrinology 92, 454-462. 33. O'Riordan, J. L. H., Woodhead, J. S., Addison, G. M., Keutmann, H. T., & Potts, J. T., Jr. (1972) "Localization of Antigenic Sites in Parathyroid Hormone," in Endocrinology 1971: Proceedings of the Third International Symposium ed. Taylor, S. (Heinemann, London), pp. 386-392. 34. Andreatta, R. M., Hartmann, A., Johl, A., Kamber, B., Maier, R., Riniker, B., Rittel, W. & Sieber, P. (1973) Helv. Chim. Acta 56, 470-473. 35. Arnaud, C. D., Brewer, H. B., Rittel, W., Bordier, P. & Goldsmith, R. S. (1973) The Endocrine Society, 55th Annual Meeting, Chicago, 1973, Abstract no. 222.
