Int. J . PeptideProtein Rex 20,1982,429-433

Synthesis of human p-endorphinyl-thiolglycine and its use for the preparation of affinity columns of p-endorphin

JAMES BLAKE, KATHERINE K. HINES and CHOH HA0 LI*

Hormone Research Laboratory, University of California, San Francisco, CA, USA

Received 30 April, accepted for publication 11 May 1982

The peptide human p-endorphinyl-thiolglycine (I) has been synthesized by the solid-phase method. The citraconyl derivative of peptide I was coupled to aminohexyl-Sepharose by reaction with silver nitrate/N-hydroxysuccinimide in water. The citraconyl groups were removed in aqueous acetic acid and the resulting resin was used in affinity chromatography for the purification of anti- sera to P-lipotropin and /3-endorphin. Key words: affinity chromatography; antiserum; p-lipotropin; coupling agent; radioimmuno-

assay ; silver nitrate/N-hydrox ysuccinimide.

Previous reports (1 -3) from this laboratory have demonstrated that water-soluble peptides containing a C- terminal thiolcarboxyl group could be selectively coupled to amino groups to form amide (peptide) bonds. Of special interest was the synthesis of a derivative of cortico- tropin, corticotropinyl-thiolglycine, which was selectively coupled at its C-terminus to bovine serum albumin (3). In order to extend this methodology to the study of the chemistry of 0-endorphin (4, 5), we have synthesized o h - endorphinyl-thiolglycine (see Fig. 1) for the preparation of an affinity column. The use of the affinity column for the purification of antisera to Ph-LPH and Ph-EP was also carried

out. Results of these studies are reported herein.

Tyr-Gly-Gly-Phe-Met - - - - - - - - - - Glu-OH 59 8 9

,< -LPH

H-Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser- 1 1 0

Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-Lys-Asn- 11 20

A l a - I l e - I l e - L y s - A s n - A i a - T y r - L y s - L y s - G l y - G l u - G l y - S H 21 31

Ph-EP-Gly-SH Abbreviations: ph-EP, human 0-endorphin; Ph-LPH, human 0-lipotropin; Gly-S, thiolglycine; Cit, citra- conyl; PSB, phosphatesaline buffer.

*Address for reprints.

FIGURE 1 The amino acid sequence of ph-LPH and PhendOI- phinyl-thiolglycine. The sequences of ph-LPH-(59- 89) and ph-EP are identical.

0367-8377/82/100429-05 %02.00/0 0 1982 Munksgaard, Copenhagen 429

J . Blake el al.

EXPERIMENTAL PROCEDURES

Protected peptide resin corresponding to p,-EP-

Boc-Gly-S-resin (1.20g, 0.42 mmol) was pre- pared as described (2) and subjected to the following procedure: (a) washing with methylene chloride, 4 times; (b) washing with 55% trifluoroacetic acid/methylene chloride; (c) reaction with 55% trifluoroacetic acid/ methylene chloride for 15 min; (d) washing with methylene chloride, 2 times; (e) washing with 25% dioxane/methylene chloride, 3 times; (0 repeat step d; (g) reaction with 5% diiso- propylethylamine/methylene chloride for 2 min; (h) repeat step d ; (i) repeat step g; (j) washing with methylene chloride, 5 times, (k) reaction with 1.5 mmol symmetrical anhydride of the Boc-amino acid for 20min; (1) addition of 0.42 mmol N-methylmorpholine and con- tinued coupling for 20min; (m) washing with methylene chloride, 3 times; (n) washng with 33% ethanol/methylene chloride, 3 times.

Solvent wash volumes were 15ml at the beginning and were changed to 18ml at Asn-20. Side chain protecting groups were: Ser, benzyl; Thr, benzyl; Glu, benzyl; Lys, o-bromobenzyl- oxycarbonyl; Tyr, benzyloxycarbonyl at residue 1 and o-bromobenzyloxycarbonyl at residue 27. After the coupling of Tyr-1, the peptide resin was subjected to steps a-e, washed with ethanol, and dried.

Gly-SH

Ph-EP-GlY-SH (Z) A sample of peptide resin (0.382g, 0.0526 mmol) was treated with 0.77ml anisole and 12 ml HF (6) for 1 h at 0". The HF was evapor- ated at O", and the residue was washed with ethyl acetate. The peptide was dissolved in 5 ml 20% acetic acid, and chromatography on Sephadex (3-10 in 0.5 N acetic acid gave 107 mg crude peptide. Chromatography of the crude peptide on CM-cellulose was performed as des- cribed (7) and gave 42mg peptide. A sample (40.2mg) of the peptide was further purified by partition chromatography on Sephadex G-50 (8) in the system n-butanol:pyridine:O.l% acetic acid . (5 :3: 10) and gave 25 mg of the final product (peptide I) at R f 0.38.

Paper electrophoresis (400 V, 3 h) of peptide I at pH 3.7 and 6.7 showed ninhydrin, chlorine

positive spots at Rtys 0.51 and 0.38, respect- ively. ph-EP showed the same mobility as I in each system. Iodometric titration (2) of a sample of peptide I indicated a 75% content of thiolcarboxyl group compared to a peptide content (determined by amino acid analysis) of 80%.

Thin-layer chromatography on silica gel in the system n-butanol: pyridine: acetic acid : water (5 : 5.1 : 4) gave a spot 'at Rf 0.56. Amino acid analysis of acid and enzyme hydrolysates is shown in Table 1.

Na-Cit[Lys(fir)9.19,24,28,29 ] -flh-EP-cly-sH (11) Peptide I (29.4 mg) was dissolved in 1 ml 6 M guanidine hydrochloride/0.05 M K2P04 pH 8.0. Then 1 ml of 0.1 M K z P 0 4 (pH 8.0) was added, and the pH of the peptide solution was adjusted to 8.0. Citraconic anhydride ( 1 4 ~ 1 in loop1 dioxane) was added in four equal portions, and sodium hydroxide was added to maintain pH 8.0. The solution was chromatographed on Sephadex G-10 in 0 . 0 2 5 ~ ammonium bicar- bonate. Fractions which contained peptide

TABLE 1 Amino acid analysis of phEP-Gly-SH

Acida Enzymeb

ASP 2.06 (2)c

7.87 (8)d Thr 2.90 (3) Ser 1.88 (2) Glu Pro

Ala Val Met Ile Leu Tyr Phe LY s

Gly

3.06(3) 0.94(1) 4.00 (4) 2.07(2) 1.00 (1) 0.99(1) 1.07 (2) 2.03(2) 1.97(2) 1.95 (2) 5.1 8 (5)

1.84(2) l.OO(1) 3.65 (4) 2.05 (2) 1.12(1) 0.89 (1) 2.00 (2) 2.14(2) 2.00(2) 2.00 (2) 5.29(5)

aHydrolysis in constant-boiling HCl; 22 h at 110". bTrypsin/chymotrypsin followed by aminopeptidase M. CNumbers in parentheses are the theo- retical values. dCorresponds to sum Asn + Gln + Thr + Ser.

430

Human -endorphinyl-thioglycine

micropipet. The aliquot and the pipet (which was broken into three to four small pieces) were transferred to a small round-bottom flask. The contents were covered with constant- boiling HCl and refluxed overnight. The acid solution was evaporated to a residue that was dissolved in water, filtered, and the filtrate was evaporated to a residue that was dissolved in pH 2.2 sodium citrate buffer. A 1/20 aliquot was submitted to the amino acid analyzer and the results indicated the presence of 2.73 nmol peptide, which corresponds to 0.76 pmol/ml of settled resin bed. For the total of 5.3ml of resin bed obtained, this corresponds to 4.0pmol peptide incorporated into the resin and a coupling yield of 8 1%.

were combined, loop1 0.56M sodium bicar- bonate was added, and the solution lyophilized to give 3 1.8 mg peptide. Paper electrophoresis (pH 6.7, 400V, 2h ) showed an impurity (ca. 10%) at R y - 0 . 4 1 in addition to the main spot at - 0.54. The sample was retreated with citraconic anhydride (7pl) by the above procedure and 29.5 mg peptide I1 was isolated. Pa er electrophoresis showed a single spot at RfyS - 0.54.

0 h-EP-Gly-Sepharose o-Aminohexyl-Sepharose 4B (Sigma; 1.746 g) in a 50ml polypropylene centrifuge tube was suspended in lOml water. After standing for 20min, 20ml 2% sodium bicarbonate was added. A spatula was used for gentle stirring, and the mixture was allowed to stand for 20 min. Centrifugation (1000 r.p.m. for 30 s), followed by removal of the supernatant, gave a residue that was washed with 20 ml 2% sodium bicarbonate and six 20-ml portions of water. A solution of 27.4mg (4.90pmol) peptide I1 in 0 .5ml water was added to the washed resin which had a bed volume of ca. 6ml. The mixture was gently stirred with a spatula and allowed to stand for 20min at room tem- perature and cooled to 0". Then a solution of 64pmol N-hydroxysuccinimide in 130p1 water (neutralized to pH 7 with sodium hydroxide) was added and the mixture was gently stirred with a spatula. After 1Omin at 0", 25pl 0.4M silver nitrate was added and the mixture was stored at 0" for 30min, and at room tem- perature for 2 h . A spatula was used for occasional stirring.

The resin was washed with two 20-ml portions of 0.25M sodium cyanide, and six 20-ml portions of water. Then 6 m l water and 3 m l acetic acid was added to remove the Cit groups in the peptide, and after gentle stirring with a spatula, the mixture was stored at room temperature for 22 h. Acetic acid was separated by decantation, the resin was washed with water, and finally poured into two columns where it gave bed volumes of 2.6 and 2.7ml. The columns were further washed with water.

For analysis, 1 .O ml water was added to the column of bed volume of 2.6 ml. The resin was resuspended in the total volume, and a lOOyl aliquot was removed with a 100-p1 disposable

Radioimmunoassay Antisera to Ph-LPH and P,-EP were raised in rabbits as previously described (9, 10). Oh' LPH (1 1) and synthetic Ph-EP (12) were iodin- ated by the chloramine T method (13) with 0.2 mCi/4pg chloramine T/pg protein. Labeled hormone was separated from free '"1 by gel filtration on Sephadex LH-20 in pyridine acetate. Specific activities of the 1-hormones were typically ca. I3OpCi/pg.

Recovery of antibody activity was calculated from total binding experiments in which 0.1 ml serial dilutions of antibody were added to each reaction tube along with 10 000 c.p.m./SO p1

assay buffer (0.01 M Na2P04, 0.15 M NaCl, 0.5% bovine serum albumin, 0.01 M EDTA, pH 7.4). After incubation for 1 8 h at 4", 0.04 ml sheep antiserum to rabbit y-globulin (diluted 1 :20) and 0.05 ml normal rabbit serum (1 : 100) were added and the samples were incu- bated for an additional 18 h at 4". Then 1 ml cold phosphate-saline buffer (PSB; 0.1 5 M NaCl, 0.01 M Na2P04, pH 7.4) was added and the samples were centrifuged, decanted, and counted in a Beckman Gamma 4000 Spectro- meter. Each antibody dilution was assayed in duplicate and the results were expressed as the percentage of the total counts (initially added to the tube) that were specifically bound by the antibody.

Radioimmunoassay followed a similar pro- cedure. Thus, 0.1 ml serial dilutions of Ph-LPH or Ph-EP were added to the reaction tubes

Of [ 12' I] 'bh -LPH or [ 125 I ] -Ph -EP and 0.1 ml

43 1

J. Blake et al.

along with 0.1 ml antisera, diluted to give 30% total binding, and 10000c .pm. /50~1 of [lzsI] - Ph-LPH or [125 I]-Ph-EP. Following incubation for 24h at 4', second antibody and normal rabbit serum were added. The samgles were incubated for an additional 18 h at 4 , 1 ml of cold PSB was added, and the tubes were centri- fuged, decanted, and counted. Samples were assayed in duplicate and the results are shown in Fig. 2.

Affinity chromatography of Ph-LPH antiserum A sample of 41.Omg antiserum powder (corre- sponding to 0.5 1 ml antiserum) was dissolved in 1.Oml PSB. The solution was applied to a column (0.72 x 3.7 cm) of p,-EP-Gly-Sepharose in PSB. A flow rate of 1 m1/40min was employed for sample application. Thereafter, the column was washed with two 4.5-ml fractions of PSB and two 4.5-ml fractions of 1 M NaCl/PSB at a flow rate of ca. 30ml/h.

peptide ( w 1 FIGURE 2 (a) The displacement of ['"I]-ph-LPH from ph-LPH antiserum (0) or purified antibody (0 ) by Ph-LPH (----) 01 ph-EP (----). (B) The displacement of [ 1251]-ph-EP from ph-EP antiserum (0) or purified antibody (0 ) by Oh LPH (----) 01 ph-EP (----).

432

The eluate was monitored by reading ODzso, which showed that essentially all of the starting material had been washed through the column. The combined four fractions were dialyzed against 41. water for 3 h at room temperature, 2 1. water for 16 h at 4", 2 1. water for 2 h at room temperature. Lyophilization gave 34.5 mg powder.

A sample (2.08 mg) of powder was dissolved in 0.44 ml PSB and the binding activity toward [1251]-Ph-LPH was determined on serial dilution aliquots. Comparison of the binding curve to that obtained for the starting antiserum indi- cated 62% recovery of binding activity.

AffiniQ chromatography of Ph-EP antiserum A sample of 16.5 mg antiserum powder (corre- sponding to 0.206 ml antiserum) was dissolved in 0.5ml PSB (PH 5.1) and 1 N phosphoric acid was added to give pH 5.1. The solution was cooled in an ice bath, and applied to a column (0.47 x 1.5 cm) of o,-EP-Gly-Sepharose that had been prewashed with PSB (pH 5.1). The entire experiment was run at 4". The flow rate during sample applications was 0.5 m1/30 min. The column was then washed (flow rate ca. 0.5 m1/3 min) with four 0.5-ml fractions of PSB (PH 5.1) and three 0.5-ml fractions of 1 M NaCl/PSB (pH 5.1). The eluate was moni- tored by measuring OD,no, which typically indicated that 95-99% of the protein had been washed through. Monitoring for binding activity against radioiodinated 0-EP showed that ca. 1% of the binding activity of the starting antiserum had been washed off the column.

The column was then eluted with three 0.55-rnl fractions of 0.1 M glycine hydro- chloride (pH 2.5). The eluate was collected in test tubes containing 1 mg,BSA and 4mg Na2HP04. Additional Na2HP04 was added as necessary to increase the pH of each fraction to 7. Only the first glycine hydrochloride fraction showed substantial binding activity toward ['"I]-fih-EP - ca. 25% compared to starting an tiserum.

RESULTS AND DISCUSSION

The peptide 0-endorphinyl-thiolglycine (1) was synthesized by the solid-phase method (14) as

Human P -endorphinyl-thioglycine allowed the greatest recovery of binding activity - ca. 25%.

The use of the purified antibody for radio- immunoassay of Ph-EP or Ph-LPH gave dis- placement curves that were essentially the same as those obtained with unpurified antisera. However, the small difference in the displace- ment curves for Ph-EP were reproducible; the Bh-EP needed for 50% displacement of [12sI]- Ph-EP from the purified antibody was 1.5-2 times that required for 50% displacement from the unpurified antiserum (Fig. 2B).

described for the synthesis of other analogs of oh-EP (15). The final peptide resin was treated with HF/anisole and the product was purified by chromatography on CM-cellulose and partition chromatography on Sephadex G-50 (8). Peptide I was characterized by amino acid analysis, paper electrophoresis, thin-layer chromatography, and iodometric titration.

Reaction of peptide 1 with citraconic anhy- dride gave the citraconyl peptide, 11, which was shown to be cleanly converted back to I by overnight treatment with 20% acetic acid.

Peptide I1 was coupled to aminohexyl- Sepharose by reaction with silver nitrate/N- hydroxysuccinimide (1,2) in water. After removal of the citraconyl groups in aqueous acetic acid, the resin was analyzed for Ph-EP content by amino acid analysis of an acid hydrolysate. The results indicated that 81% of peptide I1 had been coupled to the resin.

To characterize the resin, two types of affinity chromatography were investigated. In the first case, antiserum (9) to Ph-LPH (Fig. I ) , which cross-reacts with Ph-EP (Fig. 2A), was applied to the Sepharose column. Washing with PSB allowel' recovery of 62% of the binding activity (to [12sI]-Ph-LPH) but com- pletely removed the cross reactivity to Ph-EP (Fig. 2A). That is, the antibody molecules that were specific for the Ph-EP portion of oh-LPH, residues 59-89 (see Fig. l), were retained by the column, and the antibody molecules specific for flh-LPH-(1-58) were washed out of the column. Radioimmunoassay of Ph-LPH using the purified antibody gave a displacement curve that was indistinguishable from that obtained with antiserum.

In the second case, when the antiserum (10) to Ph-EP was applied to the column of flh-EP- Gly-Sepharose, binding activity (toward [12sI]- Ph-EP) was retained, while 95-99% of protein could be washed out. In a control experiment we showed that the binding activity in &-EP antiserum was not retained by a column of aminohexyl-Sepharose and thus there was a specific interaction between antibody to Ph-EP and flh -EP-Gly-Sepharose. Several methods were investigated for eluting antibody from the affinity column; the most efficient was glycine hydrochloride, pH 2.5. We also observed that application of the antiserum to the column at pH 5.1 instead of 7.4, and working at 4",

ACKNOWLEDGMENT

We thank W. Hain and K. Hoey for technical assistance. This work was supported in part by grants from the National Institute of Health (GM-2907), National Institute of Mental Health (MH-30245) and the Hormone Research Foundation.

REFERENCES

1 . Blake, J . (1981) fnt. J. Peptide Protein Res. 17,

2 . Blake, J . & Li, C.H. (1981) Proc. Nutl. Acud. Sci.

3. Blake, J . , Hagman, J . & Ramachandran, J . (1982) Int. J. Peptide Protein Res. 20,97-101

4 . Li, C.H. & Chung, D. (1976) Proc. Nutl. Acud. Sci. US73,1145-1148

5 . Li, C.H. (1977) Arch. Biochem. Biophys. 182,

6 . Sakakibara, S., Shimonishi, Y., Kishida, Y., Okada, M. & Sugihara, M. (1967) Bull. Chem. SOC. Jupan 40,2164-2167

7 . Li, C.H., Yamashiro, D., Tseng, L.-F. & Loh, H.H. (1977) J. Med. Chem. 20,325-328

8 . Yamashiro, D. (1980) in Hormonal Proteins und Peptides IX (Li, C.H., ed.), pp. 25-107, Academic Press, New York

9. Rao, A.J. & Li, C.H. (1977) Int. J. Peptide Protein Res. 10, 167-171

10. Chang, W.C., Yeung, H.W. & Li, C.H. (1979) Int. J. Peptide Protein Res. 13,278-281

1 1 . Li, C.H. & Chung, D. (1981) fnt. J. Peptide Protein Res. 17, 131-142

12. Li, C.H., Chung, D. & Doneen, B.A. (1976) Biochem. Biophys. Res. Commun. 72, 1542- 1547

13. Greenwood, F.C., Hunter, W.M. & Glover, J.S. (1963) Biochem. J. 89,114-123

14. Merrifield, R.B. (1963) J. Am. Chem. SOC. 85, 2 149 -21 54

15. Blake, J. , Tseng, L.-F. & Li, C.H. (1980) Int. J. PeptideProtein Res. 15, 167-170

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