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Chemical Determination of Polypeptide Hormones

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Chemical Determination of Polypeptide Hormones Proc. Nati. Acad. Sci. USA Vol. 75, No. 9, pp. 4115-4119, September 1978 Biochemistry Chemical determination of polypeptide hormones (peptide fragmentation analysis/COOH-terminal a-amide groups/gastrointestinal hormone determination) KAZUHIKO TATEMOTO AND VIKTOR MUTT Department of Biochemistry II, Nobel Medical Institute, Karolinska Institute, S-104 01 Stockholm, Sweden Communicated by Rolf Luft, May 25, 1978 ABSTRACT The presence or absence of peptide hormones the characteristic COOH-terminal amides of these hormones in tissue extracts may in certain cases be demonstrated by ex- and then dansylating the degradation mixtures and isolating posing the extracts to conditions under which characteristic the dansyl derivatives of the amides from the other prod- fragments of the polypeptide molecule in question are formed and then analyzing for such fragments. An approximate quan- ucts. titation of the hormones may also be achieved thereby. In the We demonstrate how this analytical methodology may be present work the COOH-terminal fragments of polypeptides used to follow the purification of a polypeptide hormone and containing characteristic a-amide groups were released en- to reveal the occurrence in tissue extracts of peptides with zymically and then converted into the fluorescent dansyl de- COOH-terminal a-amide structures. rivatives, which were identified by thin-layer chromatography. In this way the presence of secretin, cholecystokinin, and the vasoactive intestinal peptide in concentrates of porcine intes- MATERIALS AND METHODS tinal extracts were demonstrated by their COOH-terminal The gastrointes- amide fragments: valine (or leucylvaline) amide, phenylalanine Peptide and Enzyme Preparations. pure amide, and asparagine (or leucylasparagine) amide, respectively. tinal polypeptide hormones, secretin, cholecystokinin, and VIP, The analytical methodology used in the present study may also were prepared from porcine intestinal extracts according to be useful in devising simple and reliable chemical assay meth- Jorpes and Mutt (7), Mutt and Jorpes (8), and Said and Mutt (9), ods for the isolation of already known polypeptides and in the respectively. The concentrates of thermostable intestinal pep- isolation of previously uncharacterized polypeptides from tides, the methanol-soluble fraction, and the methanol-insoluble natural sources. fraction were prepared as described by Mutt (10). Polypeptide hormones in tissue extracts are usually determined The following enzyme preparations were used: subtilisins by either bioassay or radioimmunoassay. In view of the inter- (EC 3.4.21.14; subtilisin BPN' and subtilisin Carlsberg, Sigma actions between hormones in vivo, it is increasingly evident that Chemical Co.), thermolysin (EC 3.4.24.4; Daiwa Kasei K.K., bioassays of tissue extracts, which often contain several different Japan), chymotrypsin (EC 3.4.21.1; TLCK-treated, a-chy- hormones, may give ambiguous results about the presence and motrypsin, Merk A.G.), trypsin (EC 3.4.21.4; TPCK-treated, the concentrations of a specific hormone. Radioinimunoassay Worthington Biochem. Co.), and elastase (EC 3.4.21.11; too may be complicated by the problem of crossreactivity. Worthington Biochem. Co.). The determination of polypeptide hormones by chemical Other Materials. Dansyl chloride (1-dimethylamino- means has been, in most cases, unsuccessful in spite of the im- naphthalene-5-sulfonyl chloride) was purchased from Pierce portance of chemical assays in other hormone determinations. Chemical Co. Dansyl amino acids were obtained from Cal- In a few cases it has been possible to use chromatographic biochem. A.G. Dansyl amino acid a-amides and dansyl peptide methods of high resolution for direct physicochemical deter- a-amides were prepared from the corresponding amides by the mination of the intact polypeptide hormones (1, 2), but often reaction with dansyl chloride (11). Polyamide thin-layer sheets the peptide patterns of the extracts are so complex that this were purchased from Cheng Chin Trading Co., LTD., Taipei, approach may not be generally applicable. As pointed out Taiwan. The anion exchange resin Dowex 1 X 2 analytical previously (3, 4), it may, however, in certain cases be possible grade was obtained from Bio-Rad Laboratories, U.S.A. All to submit hormone-containing peptide mixtures to selective glassware used for the quantitation of the dansyl derivatives was fragmentation procedures which produce characteristic frag- pretreated with dichlorodimethylsilan in toluene (10%, vol/vol) ments of known hormones and to analyze the characteristic and rinsed with water and acetone before use. fragments instead of whole polypeptide molecules. For many Enzymatic Fragmentation and Dansylation of Peptides. polypeptide hormones, such characteristic fragments could be A crude or purified peptide sample, containing 0.1-10 nmol (or include) the COOH-terminal amino acid a-amides which of polypeptide a-amide (sample size usually not exceeding 1 occur in such diverse hormonal polypeptides as a-melanotropin, mg), was introduced into a siliconized glass test tube (4 mm calcitonin, cholecystokinin, gastrin, gonadoliberin, oxytocin, inner diameter X 50 mm length). The sample was dissolved in the pancreatic hexatriacontapeptide, secretin, substance P, 20 gl of a suitable buffer containing 40-200 Mug of the proteolytic thyroliberin, the vasoactive intestinal peptide (VIP), vasopressin, enzyme. The buffer used in the present study was either 0.2 M and others (5, 6). sodium bicarbonate/carbonate pH 9-10 or 50 mM 4-(2-hy- In this paper we show that the presence of secretin, chole- droxyethyl)-l-piperazineethanesulfonic acid (Hepes), pH cystokinin, and VIP in peptide concentrates from porcine in- 7-8/10 mM Ca2+. The reaction tubes were sealed with Parafilm testinal extracts may be demonstrated by exposing the con- and incubated at 370 for 4-20 hr or at room temperature for centrates to conditions of enzymatic degradation that release 24-72 hr. The degradation products were lyophilized and then redissolved in 20 Ml of 0.2 M sodium bicarbonate/carbonate, The publication costs of this article were defrayed in part by page and 20 Ml of dansyl chloride in acetone (5-20 mg/ml) was charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviations: VIP, vasoactive intestinal peptide; Hepes, 4-(2-hy- this fact. droxyethyl)-l-piperazineethanesulfonic acid. 4115 Downloaded by guest on September 26, 2021 4116 Biochemistry: Tatemoto and Mutt Proc. Natl. Acad. Sci. USA 75 (1978) added. The concentration of the dansyl chloride solution added RESULTS was chosen according to the approximately estimated content Separation and Identification of Amides as Dansyl De- of groups reactive to dansyl chloride in the sample. Initial pH rivatives. Amino acid a-amides and peptide a-amides were of the buffer was selected to maintain pH of the solution at separated from free amino acids and other peptides by dansy- 9.5-10 during the dansyl reaction. lation of the mixtures followed by extraction with ethyl acetate The reaction was allowed to proceed for 1 hr at 370 in the and thin-layer chromatography on polyamide sheets of the dark. Then 5 ,l of pyridine (spectroscopic grade) was added materials extracted into the organic phase. Provided the ex- to remove traces of free dansyl chloride that had remained in traction is carried out under alkaline conditions, the dansyl the solution. After a further 15 min of incubation at 370, the derivatives that contain no free carboxyl groups are preferen- mixture was dried under reduced pressure. To the tially extracted into the organic phase while those with free reaction carboxyl groups largely remain in aqueous solution. Conse- dried residue was added 100 tl of the aqueous phase of an ethyl quently, the dansyl derivatives of amino acids and peptides are acetate/water mixture, followed by 50 Ml of the organic phase. retained and those of amino acid and peptide a-amides ex- The two phases were then mixed vigorously on a Vortex mixer tracted. Exceptions are the dansyl derivatives of arginine amide and the mixed phases were separated by centrifugation in a and of arginine-containing peptide a-amides. The didansyl clinical centrifuge. The organic phase was carefully transferred derivatives of histidine, lysine, and tyrosine amides are ex- to another small test tube and the aqueous phase was re-ex- tracted, whereas their monodansyl derivatives are largely re- tracted with two further 5S,-l portions of the organic phase. tained. The dansyl derivatives in the organic phase were pu- The extracts were combined and evaporated to dryness. rified further by thin-layer chromatography on polyamide For application to the thin-layer sheet, the residue was dis- sheets. The chromatography was carried out in the hydrophobic solved in 10-20 ,l of a mixture of 0.2 M sodium bicarbonate/ solvent systems, pyridine/heptane (30:70, vol/vol) in the first carbonate, pH 9.5 and ethanol (1:1, vol/vol). One to 5 ,l of the dimension and chloroform/heptane (70:30, vol/vol) in the solution was applied to the sheet. second dimension. This chromatography also served to identify Thin-Layer Chromatography on Polyamide Layer. The the a-amides (Fig. 1). sample containing dansyl derivative(s) of the a-amide(s) was For application to the chromatographic sheet the dansyl applied on a 7.5 X 7.5-cm polyamide layer sheet (washed derivatives were dissolved in an ethanolic alkaline buffer. The overnight in chloroform) in
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