Peptide,s, Vol. 6, pp. 17-22, 1985. *:' Ankho International Inc. Printed in the U.S.A. 0196-9781/85 $3.00 + .00 Oxidation/Reduction of Methionine Residues in CCK: A Study by Radioimmunoassay and Isocratic Reverse Phase High Pressure Liquid Chromatography A. J. BACARESE-HAMILTON, T. E. ADRIAN, P. CHOHAN, T. ANTONY AND S. R. BLOOM 1 Royal Postgraduate Medical School, Du Cane Road, London W12 OHS, UK Received 31 May 1984 BACARESE-HAMILTON, A. J., T. E. ADRIAN, P. CHOHAN, T. ANTONY AND S. R. BLOOM. Oxidationh'eduction of methionine residues in CCK: A study by radioimmunoassay and isocratic reverse phase high pressure liquid chromatography. PEPTIDES 6(1) 17-22, 1985.--The study was undertaken to investigate the oxidation and reduction of cholecystokinin (CCK) both as pure standards and as endogenous porcine peptides. Furthermore an attempt was made to prevent oxidation of the endogenous porcine peptides in the extraction procedure. CCK-8 and CCK-33 standards were always oxidized in weak solutions, CCK-8 varying from 26% to 67% oxidized and CCK-33 from 18% to 70%. Similarly, tissue extracts of porcine brain and duodenum contained oxidized forms of the peptide. CCK standards were readily oxidized in the presence of hydrogen peroxide. Oxidized CCK-8 standard and CCK-8 in porcine brain was 90% reduced and oxidized CCK-33 standard and in duodenal extracts was reduced by 70~ by a 40 hour incubation with 0.725 mol/1 dithiothreitol at 37°C. Extraction of CCK peptides in the presence of 65 mmol/l dithiothreitol resulted in almost complete prevention of oxidation with over 95% of the peptides being obtained in the reduced state. This additive is therefore recommended for all tissue quantitation studies. Cholecystokinin (CCK) Specific radioimmunoassay Isocratic reverse phase high pressure liquid chromatography IHPLC) Dithiothreitol Peptide oxidation Methionine sulfoxide THE multiplicity of molecular forms of cholecystokinin (CCK) of varying amino acid chain lengths found in CCK- containing tissues is a well established fact [12, 14, 17]; the suifation of the tyrosine residue at position 7 from the C-terminus and the amidation of the C-terminal phenylalanine residue is also well recognized [12,16]. How- ever, the literature contains only a few reports of the possi- ble oxidation of methionine residues in CCK, which are in positions 9, 28 and 31 of CCK-33 [1, 11, 13]. The degree of oxidation of these residues has only become apparent with the advent of high resolution reverse phase high pressure liquid chromatography (HPLC). There are no reports on the reduction of the oxidized methionine residues of CCK. The amino acids most prone to oxidation are, in approx- imate order of descending reactivity: cysteine> methionine> tryptophan> tyrosine> histidine. CCK-33 con- tains three methionines, one tryptophan, one tyrosine and one histidine residue; of these tyrosine is sulfated and as such is very unlikely to be oxidized and the tryptophan and histidine residues require more rigorous oxidation conditions than are normally met in procedures used in extraction and handling for radioimmunoassay. Consequently, as oxidation of methionines is the only likely problem, only these residues have been addressed in this study. The oxidation products of methionine are as follows: ~Requests for reprints should be addressed to Professor S. R. Bloom, Department of Medicine, Royal Postgraduate Medical School, Du Cane Road. London W12 OHS UK. 17
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`18 BACARESE-HAMILTON El" AL. CH:~ J S H,.O2 r (CH,_,)~ J H-C-NHe Photo I COOH oxidation Methionine CH:~ i S-O i ICH._,k, I H-C-NH_, I COOH Methionine sulfoxide pertormic acid CH:~ I O=S O I (C H_,)., H-C-N H,, J COOH Methionine sulfone The methionine sulfone would not be produced except under very strong oxidizing conditions but the reaction is irrevers- ible. Photo-oxidation in aqueous solutions exposed to air and light will readily oxidize methionine to the sulfoxide, which is a highly polar residue and alters the non-polar characteris- tic of the side chain thereby interfering with (or even destroy- ing) biological activity. Oxidizing conditions as used in con- ventional iodination techniques, e.g., chloramine T [9] will also form methionine sulfoxide. To date this problem in the iodination reaction has been overcome by either using con- jugation iodination techniques such as the Bolton and Hunter reaction [15] (which produces biologically active tracers) or by the synthesis of peptides in which the methionine residues are replaced by threonine and/or norleucine [7]. The extrac- tion of peptides from tissues and subsequent long term stor- age provides another risk of methionine oxidation. Since proteins [2], e.g., pepsin, ribonuclease, alpha-chymotrypsin, and peptides [2], e.g., ACTH, met-leu-phe have been shown to lose biological activity by oxidation of methionine it is important to investigate the oxidation of CCK, its reversal and, if possible, its prevention. This was the aim of the pres- ent study. METHOD Peptides Pure porcine CCK-33 was a gift from Professor Mutt (Karolinska Institute, Stockholm, Sweden) and synthetic sulfated CCK-8 was kindly donated by E. R. Squibb and Sons (London). Radioimmunoassays. Two immunoassays were em- ployed; one was specific for the CCK octapeptide (CCK 26--33) and the other was specific for CCK forms larger than the octapeptide (both assays submitted for publication). Briefly, the CCK-8 specific assay (antibody OT10) was apparently specific for the sulphated octapeptide, not react- ing with non-sulfated CCK-8, large CCK molecular forms or the gastrins. The assay used an extended CCK-8 peptide which has a non-sulfated tyrosine in place of the amide group at the C-terminus (abbreviated to TYROC, for tyrosinated octapeptide) iodinated by the chloramine T technique as tracer and sulfated CCK-8 as standard. The assay could dis- tinguish 0.4 fmol/tube between adjacent tubes with 95% con- fidence. The second assay employed antibody Z-91 and used pure CCK-33 as standard and CCK-33 labelled by the Bolton and Hunter technique as tracer. The assay was specific for CCK peptides larger than the octapeptide. The assay could distin- guish 2 fmol/tube between tubes with 95% confidence. Reverse phase HPLC. This was performed on a 5 /xm Techsil C 18 column (HPLC Technology Ltd) eluted isocrat- ically with 3WYb acetonitrile/water with 0.1% trifluoroacetic acid. This was increased to 50% acetonitrile/water after" 40 minutes, and this eluant run for 30 minutes. The column was subsequently re-equilibrated with 30% acetonitrile/water be- fore the elution of other samples. Prior to loading all samples were prepared on a C18 p, Bondapak cartridge (Waters Associates Inc.) eluted with 50% acetonitrile/water with 0.1% trifluoroacetic acid. After diluting to 25% acetonitrile, centrifugation (12,000 g, 5 minutes) was performed prior to columning. Tissue extracts. A neutral extract (pH 6.5) of porcine temporal cortex was chosen as a rich CCK-8 source and an acid extract (pH 2.5) of porcine duodenal mucosa as a CCK-33 source. Both extracts were prepared as previously described in detail [3]. The weighed tissues were thoroughly diced and plunged into polypropylene tubes containing boil- ing distilled water. After a 10 minute boil the tubes were removed from the water bath, an aliquot was taken to pro- vide the neutral extract {pH 6.5). In the case of duodenal tissue, the neutral extract was acidified to a final concentra- tion of 0.5 M acetic acid by addition of 30/zl/ml of glacial acetic acid. This was re-boiled for 15 minutes and this pro- vided the acid extract (pH 2.5). Oxidation o[('('K-8 standard. This was performed as de- scribed by Dedman eta/. [4]. Standard (10 pmol) dissolved in 0.1 M sodium bicarbonate (pH 8.0) was treated with 0.1 M hydrogen peroxide in three 0.01 ml portions at 15 minute intervals. The reaction was terminated by the addition of 2 M acetic acid. One aliquot of peptide was reacted with I M hydrogen peroxide for 45 minutes and the reaction then ter- minated with acetic acid. All samples were washed through a SEP-PAK, as above, and loaded on the C18 column and eluted with 30% acetonitrile/water with 0. I% trifluoroacetic acid. One ml fractions were collected and subjected to radioimmunoassay. Oxidation of pure porcine CCK-33 standard was per- formed by an identical protocol to that of CCK-8 (above) but the standard was dissolved in 0.5 M acetic acid (pH 2.5). The elution profiles of the unoxidized standards, chemi- cally oxidized standards and of the endogenous CCK-forms from the tissue extracts were compared. Areas under the peaks were calculated to obtain percentages of oxidized and reduced forms. Reduction of Oxidized Standards and l'Lssues Oxidized standards and tissue extracts were incubated with dithiothreitol (0.725 mol/I) at 37 ° for 20, 40 and 90 hours. One half of the original tissue (both brain and gut) was extracted in the presence of 65 mmol/l dithiothreitol (1%). Dithiothreitol was added to distilled water and the extraction
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`METHIONINE RESIDUES IN CCK 19 500- 250- _ 500- o 250- .,,-~ 0 c6 tH 0- 0 E 500- I 0 o 250 - O~ 500- 250- A [, nA I B r-i i c [ f O A K I O- I i-~ 1 10 20 minutes FIG. 1. Sequential oxidation of CCK-8 standard (courtesy of Squibb and Sons) with hydrogen peroxide. CCK-8 standard was oxidized with hydrogen peroxide and after washing through a SEP-PAK (see text) the sample was loaded on a 5 p~m Techsil C18 column (HPLC Technology) and eluted isocratically with 3(Y~ acetonitrile/water with 0.1% trifluoroacetic acid. Flow rate was 1 ml/min and 1 ml fractions collected and subjected to radioimmunoassay employing the CCK-8 specific antibody OTI0 (see text). Panel (A) CCK-8 in 0.1 M sodium bicarbonate, (B) CCK-8 incubated with 0.1 M hydrogen peroxide for 15 minutes, (C) CCK-8 incubated with 0.1 M hydrogen peroxide for 30 minutes and (D) CCK-8 incubated with 1.0 M hydro- gen peroxide for 45 minutes. 500- 250- O~ 500- o 250- ¢..) F.-, ~ 0 0 E 500- to I o 250 0 500 A I B ¢] I C I I I1 D 250 1 0 15 30 minutes FIG. 2. Oxidation of pure CCK-33 (courtesy of Prof. Mutt) with hydrogen peroxide, lsocratic HPLC procedure as in Fig. I. Frac- tions were subjected to radioimmunoassay employing an antibody which is specific for CCK forms larger than the octapeptide (Z-91). Panel (A) CCK-33 in 0.5 M acetic acid, (B) CCK-33 incubated with 0. I M hydrogen peroxide for 15 minutes, (C) CCK-33 incubated with 0. I M hydrogen peroxide for 30 minutes and (D) CCK-33 incubated with 1.0 M hydrogen peroxide for 45 minutes. performed exactly as documented in the tissue extraction section of the method section. All standards and tissue ex- tracts were prepared on SEP-PAK's and eluted on the Cl8 column. Fractions were assayed by the appropriate im- munoassay. Dithiothreitol (0.1%) was included in all solu- tions, in the preparation of standards and tissue extracts and in the elution buffer of the column. RESULTS CCK octapeptide standard was always found to be oxidized, ranging from 25% to 67% (n= 12). Figure 1 shows the sequential oxidation of CCK-8 with increasing incubation times with hydrogen peroxide. As oxidation proceeded more immunoreactivity eluted at 4 minutes and 9 minutes and less at 15 minutes (retention time of the unoxidized standard). Eventually, with high hydrogen peroxide concentration (IM) most of the immunoreactivity eluted after 4 min (95%). The pattern of the oxidation of CCK-33 by hydrogen peroxide is shown in Fig. 2. CCK-33 standard, like CCK-8, was also found to be oxidized in weak solutions, varying from 18% to 70% oxidized (n= 10). Recoveries of standards from HPLC columns were greater than 90% in all cases indi- cating that cross-reaction of the antibodies with oxidized forms of the peptide must be close to 100%. Extracts of porcine temporal cortex eluted as 2 peaks as measured with a CCK-8 specific antibody. These peaks coincided with the retention times of the oxidized CCK-8 standard (Fig. 3).
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`20 BACARESE-HAMILTON ET AL. 0 g 0 I o o CCK-8 STANDARD BRAIN EXTRACT 400 10 0 -= i [~ I 0 [~ I I I 2001 200 O'J ' ~ ] O [ IJ] I 200 1 200 O-J I ~ ~ I O'J I I'll I 0o o oot ! 2001 200 OJ I I"1 r I O"J V I 0 10 2 0 0 10 210 minutes FIG. 3. Reduction of CCK-8 standard (left hand side) and of a neu- tral extract (pH 6.5) of porcine brain (right hand side) with dithio- threitol. The standards and brain extracts were incubated with 0.725 mol/l dithiothreitol at 37°C for varying lengths of time then SEP- PAKed prior to loading on the isocratic reverse phase HPLC system (as in text). Fractions were assayed using antibody OTI0. Panel IA) standard and extract in neutral solution, (B) incubation with 0.725 mol/I dithiothreitol at 37°C for 20 hours, (C) incubation with I).725 mol/l dithiothreitol at 37°C for 40 hours and (D) incubation with 0.725 mol/1 dithiothreitol for 37°C for 90 hours. 0 .r.4 o ¢H 0 I o o CCK-33 STANDARD .oo1, H'°°7 7 f :l,Oo O"J I I I O~ I 400 1 20 I I c 7 DUODENAL EXTRACT I I I I I I 0 15 30 0 15 30 minutes FIG. 4. Reduction of pure CCK-33 standard (left hand side) and of an acid extract (pH 2.5) of porcine duodenum (right hand side) with dithiothreitoh All details as for Fig. 3. Fractions were assayed with antibody Z-91. Panel (A) standard and duodenal extract in acid solu- tion, other panels show incubation of standard and duodenal ex- tracts with 0.725 mol/I dithiothreitol at 37°C for 20 hours IB), 40 hours {CI and 90 hours ID). Porcine duodenal mucosa extracts eluted in various peaks some of them coinciding with the retention times of the oxidized pure CCK-33 standard (Fig. 4). Reduction of oxidized CCK-8 standard was attained by a 40 hour incubation with dithiothreitol (0.725 tool/l) at 37°C. A similar incubation resulted in more than 95% of endogenous CCK-8 in porcine cortex being reduced (Fig. 3). Reduction of oxidized CCK-33 required more rigorous conditions: 70% reduction was achieved after a 40 hour incu- bation at 37°C with dithiothreitol. In a porcine duodenal extract total reduction was not obtained even under these conditions (Fig. 4). A 90 hour incubation with dithiothreitol at 37°C resulted in peptide of both pure CCK-33 standard and of the duodenal extract eluting as a large immunoreactive peak after 6 minutes. Extraction of endogenous CCK's in the presence of dithiothreitol produced elution profiles for por- cine brain and duodenal tissue as seen in Fig. 5. Most of the immunoreactivity eluted in the position of the reduced standards. DISCUSSION The HPLC profiles demonstrate that oxidation of the methionines in CCK is readily achieved and that the oxida- tion products of CCK are more polar than the reduced forms, i.e., elute earlier from columns discriminating on a hydrophobic basis. It is also apparent that CCK-33 is more resistant to reduction than CCK-8. Porcine cortex is a good source of CCK-8 as more than 90% of the endogenous CCK is in this molecular form [5] but duodenal mucosa contains both larger and smaller CCK forms than CCK-33 [10], presumably which are also oxidizable. In the extracts of duodenum chosen, the major molecular forms detected by the antibody employed (Z-91) are CCK-58, CCK-39, CCK-33 and a molecular form eluting between CCK-8 and CCK-33 standards. At 30% acetonitrile CCK-58 is retained on the col- umn, so the only molecular forms eluting are CCK-33 and 39 and the form eluting between CCK-8 and CCK-33. The latter form has a shorter retention time than CCK-33 on this system
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`METHIONINE RESIDUES IN CCK 21 500- 250- O O O- G E 500- P-4 I r...? 250- O_ CCK-8 CCK-33 I I • I 0 15 30 minutes FIG. 5. Extraction of porcine immunoreactive CCK in the presence of 65 mmol/I dithiothreitol. Extraction and HPLC details as in text. Upper panel: porcine duodenal extract (pH 2.5) as measured by antibody Z-91, lower panel: porcine brain extract (pH 6.5) as meas- ured by antibody OT10. The retention limes of the pure CCK sland- ards are indicated. (unpublished, personal observation). Endogenous peptides ap- pear to be largely unoxidized as extraction in the presence of dithiothreitol results in over 90% of the peptides being in the reduced state. Reduction of methionine and of methionine in a model heptapeptide have been considered most comprehensively by Houghten and Li [8]. The reducing agent of choice must not modify the peptide in any other fashion, e.g., by acetyla- tion, and the incubation conditions should avoid degradation of the peptide, e.g., by deamidation. Dithiothreitol, a thiol, was the reagent chosen for this particular study. Reduction of CCK-octapeptide was successful both in standard and tis- sue extracts. The reduction of CCK-33 was not complete particularly in tissue extracts, and the incubation conditions employed would be expected to deamidate the peptide. Modification to the peptide was evident in Fig. 4 (panel D) in which a large immunoreactive peak eluted after 6 minutes, this may be due to bacterial contamination. The tissue also contains CCK-peptides other than CCK-33 and this compli- cates interpretation of the data. Once oxidized the reduction of methionine residues within a peptide is difficult to accomplish. It is preferable to prevent the oxidation of the peptide in the extraction medium. Our preliminary study on the extraction of CCK peptides in the presence of dithiothreitol suggests that this reagent should be added for all tissue quantitation studies. The oxidation of molecular forms of peptides is an impor- tant issue--not only can oxidized molecular forms be sepa- rated from their reduced counter parts (Dockray's second CCK-8 like peptide in sheep brain [6] as characterized by ion-exchange chromatography) but pathological conditions may involve oxidation of methionine in proteins with con- current loss of biological activity, e.g., emphysema, rheumatoid arthritis and cataracts [2]. Further studies are essential on other methionine-containing peptides but our results on CCK indicate that extraction of these peptides must be performed in the presence of dithiothreitol (or a similar reducing agent) to overcome the problem of oxidation of the molecular form(s). REFERENCES 1. Beinfeld, M. C., R. T. Jensen and M. J. Brownstein. HPLC separation of cholecystokinin peptides. Two Systems. J Liquid ('hromatogr 3: 1367, 1980. 2. Brot. N. and H. Weissbach. The biochemistry of methionine sulphoxide residues in proteins. 7rend~ Biol Sci 7: 137-139, 1982. 3. Bryant, M. G. and S. R. Bloom. Measurement in tissues. In: Radioimmammssay ~['Gut Regulatory Peptides. edited by S. R. Bloom and R. G. Long. London: Saunders Co. Ltd., 1982, pp. 36-41. 4. Dedman, M., T. Farmer and C. Morris. Oxidation-reduction of corticotropin. Biochem .I 66: 166-177, 1957. 5. Dockray, G. lmmunochemical evidence of CCK-like peptide in brain. Nature 264: 568-570, 1976. 6. Dockray, G., R. Gregory, J. Hutchinson, J. Harris and M. Runswick. Isolation, structure and biological activity of two cholecystokinin octapeptides from sheep brain. Nature 274: 711-713, 1978. 7. Fourmy, F., L. Pradayrol, J. Antoniotti, P. Esteve and A. Ribet. Purification of radio-iodinated cholecystokinin peptides by reverse phase HPLC. J Liquid Chromatogr 5: 757-766, 1982. 8. Houghten, R. and C. H. Li. Reduction of sulphoxides in pep- tides and proteins. Anal Biochern 98: 36-46, 1979. 9. Hunter, W. M. and F. C. Greenwood. Preparation of iodine-131 labelled human growth hormone of high specific activity. Na- tare 194: 495-496, 1962. 10. Jansen, J. and C. Lamers. Radioimmunoassay of cholecystoki- nin in human tissue and plasma. Clin Chim Acta 131: 305-316, 1983. 11. Mutt, V. Behaviour of secretin, CCK and PZ to oxidation with hydrogen peroxide. Acta Chem Stand (B) 18: 2185-2186, 1964. 12. Mutt, V. Chemistry of the cholecystokinins. In: Gastrins and the Vagus, edited by J. Rehfeld and E. Amdrup. London: Aca- demic Press, 1979, pp. 57-71.
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`22 BACARESE-HAMILTON ET AL. 13. Pradayrol, L., N. Vaysse, J. Cassigneul and A. Ribet. Use of reverse phase high performance liquid chromatography for purification and purity control of somatostatin and CCK-PZ family peptides. In: Hormone Receptors in Digestion and Nu- trition, edited by G. Rosselin, P. Fromageot and S. Bonfils. Amsterdam: Elsevier/North Holland Biomedical Press, 1979, p. 95. 14. Rehfeld, J. Four basic characteristics of the gastrin- cholecystokinin system. Am J Physiol 240: G255-266, 1981. 15. Rehfeld, J. F. Immunochemical studies on cholecystokinin. I. Development of sequence specific radioimmunoassays for por- cine triacontatriapeptide cholecystokinin. J Biol Chem 254: 4016-4021, 1978. 16. Rubin, B. and S. Engel. Some biological characteristics of cholecystokinin (CCK/PZ) and synthetic analogs. In: Frontiers in Gastrointestinal Hormone Research, edited by J. Anderson. Stockholm: Almquist and Wiskell, 1973, pp. 44-,55. 17. Straus, E. and R. Yalow. Species specificity of cholecystokinin in gut and brain of several mammalian species. Pro¢' Natl Acad Sci USA 75: 486-489, 1978.
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