A method for preparing β-hCG COOH peptide-carrier conjugates of predictable composition

Molecular Immunology, Vol. 17, pp. 749-756. ~Pcrgatnon PressLtd. 1980. Prinlcd in Great

0161-S890/80/0601~749

SOZ.OO/O

Britain.

A METHOD FOR PREPARING P-hCG COOH PEPTIDE-CARRIER CONJUGATES OF PREDICTABLE COMPOSITION ARTHUR

C. J. LEE,’ JOHN E. POWELL,’ GEOFFREY W. TREGEAR,Z HUGH D. NIALL* and VERNON C. STEVENS’

‘Department of Obstetrics and Gynecology, Ohio State University, Columbus, Ohio, U.S.A. and 2Howard Florey Institute, University of Melbourne, Melbourne,Austraha (First received 24 August 1979; in revisedform

14 November 1979)

Abstract-A

method for coupling peptides related to the beta subunit of human chorionic gonadotropin (hCG) to macromolecular carriers that permits covalent conjugation in a predictable fashion was developed. Peptides representing hCG beta subunit residues 109-145 and 11I-145 were coupled to tetanus toxoid, flagellin, synthetic polypeptides and Ficoll. Carrier compounds containing amino groups but devoid of sulfbydryl groups were reacted with 6-maleimido caproic acyl N-hydroxy succinimide ester (MCS) under conditions that result in the bifunctional reagent attached to carrier amino groups with the stable maleimido group free. Subsequently, peptides containing sulfhydryl groups were reacted with the reagent modified carrier whereby the peptides coupled to the carrier via the reaction between sulfhydryl groups on peptide and the maleimido group on carriers. Peptides devoid of sulfhydryl groups were thiolated using homocysteine thiolactone. The efficiency of coupling was confirmed by amino acid analysis and SDS polyacrylamide electrophoresis. Results indicated that the coupling of peptides to carriers could be regulated by the number of moles of MCS reacted with carrier to yield free maleimido groups. Sulfbydryl group reaction with maleimido groups was stoichiometric. Conjugates prepared by this method were used to immunize rabbits and significant levels of antibodies to the hCG peptides have been attained.

INTRODUCTION

The authors have endeavored to develop a vaccine against human chorionic gonadotropin (hCG)* based on the use of synthetic peptides representing the carboxyl terminal portion of the beta subunit of that hormone as an immunogen. Studies to date have indicated that antibodies generated to peptides containing 35-40 amino acid residues of the subunit are capable of reacting with intact hCG in vitro and neutralizing its hormonal activity in viva (Stevens, 1975). A major problem has been the production of high levels of anti-hCG antibodies in experimental animals following active immunization with peptide antigens. Attempts to enhance the immunogenicity of peptides by conjugating them to macromolecular carriers using classical cross-linking reagents has been only partially successful (Stevens, 1976). In

addition, random reactions of linking functional groups on peptide and carrier result in a variety of products. Varying ratios of polymerized peptide, polymerized carrier and peptide-carrier conjugate are produced from preparation to preparation. Products from these conjugation reactions are complex molecules, extremely difficult to characterize, and are not reproducible. Due to these problems, a method of conjugation was devised that permits predictable coupling of peptides to carriers. The method reported here utilizes a bifunctional reagent that under appropriate conditions can be specifically reactive to amino groups on carriers and specifically reactive to thiol groups of peptides. This procedure requires that carriers used contain few thiol groups but have an abundance of amino groups, and peptides either contain or have introduced into them a thiol group. Various modifications of the method are feasible using the basic principle of end group specificity of the bifunctional reagent.

*Abbreviations used: AHTL, N-acetyl homocysteine thiolactone; hCG, human chorionic aonadotrooin; DMF. dimethylformamide; DTT, dithioth;eitol; DTBA, 5,5’dithio-2-nitrobenzoic acid: MCS, 6-maleimido caproic acid MATERIALS AND METHODS N-hydroxylsuccinimide ester; NEM, N-ethyl maleimide; Peptides TGAL, poly (r_-Tyr,L-Glu)-poly(o,L-Ala,L-Lys); DL-AL, pofy(D,L-ALa,L-Lys); TT, tetanus toxoid; AECM Ficoll, 2Peptides representing the C-terminal residues amino ethyl carbamyl methylene Ficoll; SDS, sodium dodecyl sulfonate. of hCG-/I subunit 11l-145 and 109-145 were 749

750

ARTHUR

synthesized by a solid-phase peptide synthesis procedure (Tregear et al, 1977). The 111-14.5 peptide contained no cysteine, while peptide 109-145 has cysteine at the 110 residue. Detailed description of these syntheses will be presented elsewhere (G. W. Tregear, in preparation). Carriers used Tetanus toxoid (TT) was obtained from the Connaught Laboratories, Willowdale, Ontario, Canada. This preparation containing 2215 Lf units per mg of N, was further purified by gel filtration (Bio-Gel P-60). Void volume protein contained 60 equivalents of amino groups per lo5 daltons and 0.5 equivalents per 10s daltons of thiol groups. Polyacrylamide electrophoresis at pH 8.5 using 7% acrylamide gel showed more than three components. Flagellin (prepared from Salmonella typhimurium by Dr. Christopher Parish, Canberra, Australia), poly(L-tyr,L-glu)poly(D-L-ala, L-lys)[TGAL) average mol. wt of 1.1 x 10s], and poly(D-L-ala&-lys) [poly D-L-AL, average mol. wt of 6 x 104] were used as supplied. The synthetic polypeptides were a generous gift of the Weizman Institute of Science, Rehovot, Israel. The amino group content of flagellin, TGAL and poly D-L-AL was 40, 130 and 170 equivalents per lo5 daltons of weight, respectively. Ficoll,,,, sucrose polymerized with epichlorohydrin to an average mol. wt of 4x 105, was purchased from Pharmacia Fine Chemicals, Piscataway, NJ, U.S.A. Chemicals and reagents The bifunctional conjugating reagent, 6-maleimido caproic acyl N-hydroxy succinimide ester (MCS) was synthesized according to the method of Keller & Rudinger (1975) by Dr. Robert Coombs (Sandoz-Wander, East Hanover, NJ, U.S.A.). Thiol groups were introduced into peptide 111-145 at the Nterminus and at the e-amino 122 Lys using Nacetyl homocysteine thiolactone (AHTL, Mann Research Laboratories, Orangeburg, NY, U.S.A.). The organic solvent used to dissolve MCS, dimethyl formamide (DMF, Eastman Organic Chemicals, Rochester, NY, U.S.A.), was purified by treatment with tosyl chloride and vacuum distillation. The purified DMF was stored over dessicant (molecular sieve 4A, Union Carbide Corp.) Dithiothreitol (DTT) used as a reducing agent was analytical grade (Pierce Chemicals, Rockford, IL, U.S.A.).

C. J. LEE et ul.

Anal_yticalmethods All protein concentration determinations were performed using the method of Folin-Ciocalteau (1927). Amino group contents of carriers was evaluated by reaction with fluorescamine (Fluram, Hoffman-LaRoche, Nutley, NJ, U.S.A.) and measured in an Aminco-Bowman spectrophotofluorometer (American Instruments, Silver Springs, MD, U.S.A.). Thiol group determinations for testing completeness of reduction of peptides or the number of maleimido groups on carriers were performed using 5,5’-dithio-bis-2-nitro benzoic acid (DTBA) as a source of chromophore (Sedlack & Lindsay, 1968). For the later procedure, the consumption of 15 nm mercaptoethanol (in 10 ~1 0.1 M EDTA) was assessed. Amino acid analysis of carriers, peptides and conjugates was used as one of the parameters for estimating efficiency of conjugations. Conjugates, carriers and peptides were hydrolyzed for amino acid analysis in an evacuated, nitrogen-filled, sealed vial by hydrochloric acid at 110” C for 24 hr. Hydrolysates were analyzed on a model of 121 M Beckman amino acid analyzer. Since all peptides used were devoid of L-valine but were rich in L-proline ( 10 residues/mole of peptide), the ratio of proline to valine residues in conjugate hydrolysates, as compared to that of the carrier, was an accurate indicator of the moles of peptide conjugated to 10’ daltons of carrier. The calculation of conjugation rates using ratios of other stable amino acids were also used and findings agreed with those using proline:valine ratios. SDS-polyacrylamide gel electrophoresis, for molecular weight estimations, was performed using 5”/, polyacrylamide gel prepared in 0.5M phosphate buffer pH 7.0, containing O.S:, SDS using an Ortec Slab-Gel electrophoresis. Prior to electrophoresis the sample was incubated in O.lM sodium phosphate buffer, pH 7.0, containing 1% SDS and 1% mercaptoethanol at 40°C for 2 hr. Gels were stained with Coomassie blue and destained in 7”i, acetic acid. Preparation of MCS-modifed

carriers

The bifunctional reagent was coupled to carrier molecules in a molar ratio according to the desired number of peptides to be conjugated. Routinely, a conjugate with approximately 20 peptides per lo5 daltons of carrier was prepared. For TT, TGAL, poly D-L-AL and flagellin, 20 mg of carrier was dissolved in 1.O ml of 0.1 M sodium phosphate buffer, pH 6.66, and reacted with 6.4

Preparation

of hCG-Peptide

mg of MCS dissolved in 0.1 ml of DMF. The pH of the flagellin solution (0.01 N HCl, pH 2.0) was adjusted to pH 6.66 by the addition of 0.3M sodium phosphate buffer prior to addition of MCS. The mixture of carrier and MCS was stirred for 1 hr at 20°C. The maleimidated carriers were purified by gel filtration on Sephadex G-25 in water. Excluded materials with O.lM sodium equilibrated were phosphate-O.lM EDTA buffer, pH 6.66, and concentrated to a volume of 1.0 ml by ultrafiltration. In order to introduce amino groups into Ficoll, a quantity of Ficoll was carboxymethylated and converted to amino ethyl carbamyl methylene Ficoll (AECM-Ficoll) following the method of Inman (1975). For preparation of conjugates using AECM-Ficoll, 20 mg of a lot containing 45 amino group equivalents per lo5 daltons was dissolved in 1.O ml of 0.1M sodium phosphate buffer, pH 6.66, and reacted with 2 portions of MCS at t, and t,, mins. Each portion of MCS, dissolved in 0.1 ml DMF, contained an equivalent number of moles to the total amino groups in the carrier. The reaction was terminated at the end of 2 hr by applying the mixture to a column of Sephadex G-25 in water. The MCS-modified Ficoll was equilibrated in and stored by the buffer, concentrated, procedure described above for the protein and synthetic polypeptide carriers. Preparation of peptides

Disulfide dimers of peptide 109-145 were converted to monomer form by reduction with DTT. Typically, peptide (20 mg) and DTT (20 mg) were dissolved in 0.5 ml of O.lM sodium phosphate buffer, pH 8.0, and stirred under N, gas for 4 hr at 20°C. The peptide 11I-145, devoid of any cysteine residue, was thiolated by mixing 20 mg with a 5-fold molar excess of AHTL dissolved in 0.2 ml of N,-saturated M imidazole and stirred under N, for 18 hr at 20°C. The thiolated peptide was then reacted with DTT for 4 hr at 20°C (as done with the other peptide) to assure all peptides were monomeric. Monomeric forms of both peptides were purified by gel filtration on Sephadex G-25 (superfine) using a column 1.6 x 60 cm equilibrated with 0.2M acetic acid to prevent formation of S-S bonds. Peptides, separated from buffer salts and reagents, were lyophilized directly in the 0.2M acetic acid and the thiol groups determined. Peptides were stored under a

Conjugates

751

nitrogen atmosphere in a refrigerated dessicator. Conjugation of peptides to modiJied carrier

The modified carriers containing approximately 20 maleimido reactive groups per lo5 daltons were coupled to peptides containing a thiol group. In a typical experiment, 20 mg of a MCS-modified carrier was dissolved in 1.Oml of N,-saturated O.lM sodium phosphate-O. 1M EDTA, pH 6.66, buffer. This solution was added to a vial containing an amount of dry peptide equivalent to 1.2 times the molar equivalent of maleimido groups in the carrier. The reaction mixture was stirred under N, for at least 18 hr at 20°C. The conjugated product was separated from unreacted peptide by gel filtration through a Bio-Gel P-60 column 1.6 x 60 cm equilibrated in 0.2M NH,HCO,. The conjugate, eluted in the void volume of the column, was lyophilized. Aliquots of the lyophilized conjugate were removed for amino acid analysis and SDS polyacrylamide gel electrophoresis. Immunizations production

and

evaluations

of

antibody

Evaluations of the immunogenicity of peptide conjugates was accomplished by immunizing rabbits. Details of the immunizations, sera collection and antibody evaluations are described elsewhere (Powell et al., 1980). Briefly, 1.0 mg of conjugate was dissolved in 0.5 ml of saline emulsified with an equal volume of Complete Freund’s Adjuvant and injected intramuscularly, using 3 or 4 separate sites, into the thigh region of a 3-5 kg rabbit. Subsequent injections was made 21 and 42 days later using the same conditions. Bleeding commenced on day 21 and weekly thereafter. Sera were separated and stored at 20°C until evaluated. Levels of antibody to peptide and intact hCG were determined by reacting dilutions of sera with lz51 labelled antigens. RESULTS

Reaction of bifunctional reagent with carriers

Since the hydrolysis rate of the succinimide ester portion of MCS is quite rapid (Bolton & Hunter, 1973), MCS was reacted with carrier in excess. The efficiency of the N-succinimide ester to react with amino groups on carriers was tested by mixing increasing quantities of MCS with a fixed amount of carrier. Efficiency (moles reacted/moles added) was assessed measuring the free maleimido groups on carrier after the

ARTHUR

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C. J. LEE et crl.

70-

5 ‘6 .- 60‘= w 50.-S 2 400) a 30ae eoIO-

1 , , , , , , , 20 25 Jo 35 5 IO 15 Equivalents of Maleimido groups/t~da~s of Tetanus Toxoid

Fig. 1. The efficiency of N-hydroxy succinimide ester of MCS to couple with tetanus toxoid containing 60 amino groups per IO5 daltons. Increasing moles of MCS were incubated at pH 6.66 and the maleimido group content assessed following purification of modified carrier from unreacted reagent.

reaction. As shown in Fig. 1, MCS reacted with TT with high efficiency until a concentration of 15 MCS groups per lo5 daltons was attained. Above this concentration, a large excess of MCS had to be added in order to attain larger numbers of MCS groups to the carrier. Reaction efficiency was titrated for each carrier used so that the desired number of maleimido groups could be

obtained predictably from batch to batch of conjugate. The specificity of the maleimido portion of MCS for amino and thiol groups at various pH levels was also tested. For this purpose a model reagent, N-ethyl maleimide (NEM), was used. Reactivity of the maleimido group with mercaptoethanol(-SH) and ctN-acetyl-L-lysine (-NH,) in buffers from pH 6 to 10 represented reactivity to thiol and amino groups, respectively. In Fig. 2, it can be seen that this reagent reacted with thiol groups with lOOo/;, efficiency at pH 6.0-8.0. Also, it was found that no significant reaction with amino groups was observed at a pH below 7.2. Therefore, MCS reacted with thiol-free carriers at a pH below 7.0 did not result in any cross-linking of the carrier.

Conjugation

ofpeptides

1. The efficiency of conjugating peptides to MCS-modified protein

Conjugation description

Fig. 2. Reactivity of the maleimido-reactive group with thiol and amino groups at pH levels from 6 to 10. A model compound (N-ethylmaleimide) was reacted with either mercaptoethanol (-0) or al\r-acetyl-L-lysine (- x -) and the consumption of -SH or -NH? groups determined after 30 mm.

carriers

Table 1 illustrates the efficiency of peptide coupling to MCS-modified TT and flagellin. An excess of thiol containing peptide, relative to the number of maleimido groups on carrier, was added to the reaction mixture. As can be seen in Table 1, all of the maleimido groups on carrier were coupled with a peptide thiol group yielding conjugates with the number of peptides per lo5 daltons carrier equivalent to the original maleimido content of modified carrier. These observations confirm our study with the model compounds that the maleimido-SH reaction under appropriate conditions is thiol-specific and stoichiometric. Table 2 illustrates the conjugation efficiency of to TGAL, poly D-L-AL and peptides AECM-Ficoll. In these experiments, more moles of maleimido groups were added to carrier than the desired number of moles of peptide to be conjugated. Subsequently, the molar concentrations of peptides desired in the conjugate was reacted with the MCS-modified carrier. As

Table

PH

to MCS-modljied

111-145: TT 109-145: TT 111-145: Flagellin 109-145: Flagellin 10%145: Flagellin ’ As determined b As determined

Maleimido group content per lo5 dalton carrier”

thiol-containing carriers

Moles of peptide conjugated to lo5 dalton Carrie?

21 27 24 20 11 by reaction with DTBA. by amino acid analysis.

25 26 21 20 10

Preparation of hCG-Peptide Conjugates

753

Table 2. Efficiency of peptide coupling to synthetic macromolecule carriers where an excess of maleimido groups was present on carrier Moles of peptide per lo5 dalton carrier Conjugate 109-145 TGAL 109-145 TGAL 109-145 Ficoll,,,

Maleimido group content per lo5 dalton carrier” 3.5 31 29

Added to conjugation Resulting in mixtureb conjugated product 25 25 25.4

26’ 23-25’ 25.5’

y Molar extinction coefficient 13.6 x lo3 for 2-nitro-5-thiobenzoate in maleido group content estimation. bSH content estimation. ‘Determined by amino acid analysis. dDetermined by fluorescamine-NH, quantitation method

at 412 nm was used

MCS-Flagellin

flagellin Pep-flagellin (11/105daltons) Pep-fla ellin 3 daltons) (20/10

Ovalbumin

BSA

PhosphorylaseA

Fig. 3. Migration of flagelfin and conjugates of flagellin containing different numbers of peptides per 10” daltons on SDS-polyacrylamide gel electrophoresis.

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ARTHUR

shown in Table 2, complete coupling peptide to the carrier was found. SDS electrophoresis

of all added

of conjugates

Electrophoresis was performed with conjugates using carriers and proteins of known molecular weight as standards. Carriers that were impure, such as TT, TGAL and poly D-L-AL, revealed several bands on electrophoresis and it was impossible to show accurate increase in molecular weight after conjugation with peptides. However, when highly purified carriers were used. such as flagellin, an increase in molecular weight could be demonstrated with conjugates of increasing numbers of peptides per lo5 daltons (Fig. 3). These measurements permitted molecular weight estimations of conjugates with an accuracy $20,000 daltons or the addition of approximately 5 peptides/ lo5 daltons carrier. While this procedure was useful in visually demonstrating the quantitative aspects of the conjugating procedure, it did not permit as precise a measurement as the quantitation of maleimido residues on carrier after reaction with MCS and before conjugation to peptide. SDS gel electrophoresis was not applied to molecular weight estimations of Ficoll conjugates. Antibody

production

by conjugates

Figure 4 illustrates antibody levels to hCG and peptide 109-145 attained in a rabbit following immunization with a conjugate of peptide

(

J. LEE

et al.

109-145 and TT. Similar levels have been attained using conjugates of the other peptide and other carriers. Data from immunological studies using these conjugates will be presented elsewhere.

DISCUSSION Attempts to prepare hCG peptide-carrier conjugates using classical methods employing carbodiimides, glutaraldehyde and diisocyanates have yielded conjugates with only low numbers of peptides per 1OSdaltons carrier (unpublished). Also these reagents cause polymerization of peptide resulting in the coupling of peptide aggregates to carrier rather than peptide monomers. In addition, cross-linking of carrier molecules by these coupling reagents often reduced the immunogenicity of the carrier, thereby diminishing its helper effect to antibody production (unpublished data). It should be noted that various modifications of our procedure can be made to meet the needs of particular experimental conditions. For example, if an amino group containing peptide or other small molecule without a thiol group is to be coupled to a carrier, thiol groups can be introduced into the carrier and the small molecule reacted with MCS rather than the carrier. The MCS-modified peptide can then be via the efficient coupled to the carrier maleimido-thiol reaction. Also, if the protein

Fig. 4. Antibody levels attained to b-hCG peptide 109-145 and to hCG from immunizations with a conjugate of /3-hCG peptide 109-145 coupled to TT in a ratio of 20 peptides/105 daltons TT.

Preparation

of hCG-Peptide

selected as a carrier contains significant numbers of thiol groups, these can be oxidized to be made unreactive with maleimido groups prior to coupling MCS to carrier via the N-hydroxy succinimide ester-group reaction. Another approach to conjugation is to couple the maleimido group of MCS to a thiol containing small molecule prior to reaction with the carrier. The succinimide ester group of MCS can be reacted with amino groups on the carrier as the last step of the procedure. However, when using this approach it should be recognized that the succinimide ester group is labile to hydrolysis in aqueous solutions and the coupling efficiency is low. Therefore, this latter approach should not be used when reaction to MCS to carrier first is possible and is not recommended as a practical procedure. It is perhaps worthy of note that the more conventional methods of determining conjugation efficiency, such as increase in weight of carrier, increase in molecular size of carrier and amino acid analysis, did not appear to be as precise as the determination of maleimido groups on carrier after MCS modification. While these latter measurements are made before the actual conjugation of peptides to carrier, the highly efficient maleimido-thiol reaction assures a predictable rate of conjugation when adequate moles of SH-containing peptides are present (Table 2). Others have reported the use of similar bifunctional reagents for preparing well defined conjugates of other antigens and carriers. Rector et al. (1978) utilized the N-hydroxy succinimide ester of iodoacetic acid to couple gamma globulin to thiolated ovalbumin. Kikutani et al. N-(maleimido benzoyloxy) (1978) used succinimide ester reagent for conjugation of hCG to P-galactosidase for use as a tracer in an immunoassay. The advantages of coupling efficiency and prediction of molecular site of antigen attachment makes all of these methods attractive procedures for many types of immunological studies. The development of the procedure described in this paper has permitted the design of numerous experiments to develop the optimal immunogen for an antifertility vaccine based upon the use of synthetic peptide of hCG$ subunit. Numerous studies are currently underway in small laboratory animals and nonhuman primates for evaluation of the feasibility of this approach to the regulation of human fertility.

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Conjugates

CONCLUSIONS

The conclusions reached from this study are: (1) Peptides of the hCG beta subunit containing a sulfhydryl group can be efficiently and predictably coupled to carriers containing amino groups using the bifunctional reagent MCS; (2) The determination of free maleimido groups on the MCS-modified carrier is an accurate indicator of the rate at which sulfhydryl-containing peptides will couple to carriers; (3) Immunizations with conjugates of hCG-/I COOH peptides and tetanus toxoid prepared by MCS coupling yield high levels of antibodies to the peptide. Acknowledgements-The authors wish to thank Dr. Henry Keutman, Harvard Medical School, Boston, Massachusetts, for performing the amino acid analyses, and Susan S. C. Lee for technical assistance in preparation of the conjugate described. Our appreciation is expressed to Dr. Robert Coombs for his considerate efforts in preparing reagents and providing helpful suggestions during this study. Financial support for this study was provided by the World Health Organization.

REFERENCES Bolton A. E. &Hunter W. M. (1973) The labelling ofproteins to high specific radioactivities by conjugation to a itsIcontaining acylating agent. Biochem. J. 133, 529-538. Folin 0. & Ciccalteau V. (1927) On tyrosine and trvptophane determinations in proteins. J. bioj. Chem. 73, 627-650. Inman J. (1975) Thvmus-independent antirrens: the preparation of ‘covalent, hap&n-Ficoll conjigates. J. Itnmun. 114, 704-709. Keller 0. & Rudinger J. (1975) Preparation and some properties of maleimido acids and maleoyl derivatives of peptides. Helv. Chim. Acta 58, 531-541. Kikutani M., Ishiguro M., Kitagawa T., Imamura S. & Miura S. (1978) Enzyme immunoassay of human choriogonadotropin employing B-galactosidase as label. J. clin. Endocrin. Metab. 47, 980-984. Powell J. E., Lee A. C., Tregear G. W., Niall H. D. & Stevens V. C. (1980) J. Reprod. Immunol.. in press. Rector E. S., Schwenk R. J., Tse K. S. & Sehon A. H. (1978) A method for the preparation of protein-protein conjugates of predetermined composition. J. Zmmun. Melh. 24, 321-336. Sedlack J. & Lindsay R. H. (1968) Estimation of total protein bound and non-protein sulfhydryl groups in tissue with Ellman’s reagent. Analyr. Biochem. 25, 192-205. Stevens V. C. (1975) Antifertility effects from immunizations with intact, subunits, and fragments of hCG. In Physiological Effects of Immunity Against Reproductive Hormones (Edited by Edwards & Johnsons). Cambridge University Press, London. Stevens V. C. (1976) Actions of antisera to hCG-/I: in vitro and in vivo assessment, Excerpta Medica International Congress Series No. 402 Endocrinology. Proceedings of the V International Congress of Endocrinology, Hamburg, ._ _. _ . ..__ IX-24 July, 1976.

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Tregear G. W., van Rietschoten J.. Saur R., Niall H. D., Keutman H. & Potts J. T.. Jr. (1977) Synthesis, purification. and chemical characterization of the amino-

C. J. LEE (‘I ul termmal 1-34 fragment of bovine parathyroid hormone synthesized by the solid-phase procedure BiochcwzrJtr~ 16, 2817-282X.