Patent GB-1394846-A (GB)
Title: Polypeptides
IPC: A61P1/00
Filed: 1972-11-29
Abstract: 1394846 Fractionating urogastrone IMPERIAL CHEMICAL INDUSTRIES LTD 17 Oct 1973 [29 Nov 1972] 55089/72 Heading A5B [Also in Division C3] A process for the fractionation of crude urogastrone into three components comprises the steps of: a) applying a sample of a urogastrone preparation having a specific biological activity as defined above of 5-10 ug./kg to a column of carboxymethyl cellulose equilibrated with an 0.01 molar aqueous buffer of pH 4.5, eluting the column with a buffer of pH 5.0 with a concentration gradient of 0.01 molar to 0.5 molar, and collecting the eluted material showing the biological activity, b) applying the biologically active material from step a) to a column of a porous polyacrylamide gel excluding molecules of molecular weight greater than 5,000 in an 0.05 molar aqueous buffer solution of pH 7.2 and composed of volatile components, eluting the column with the same solution, and collecting the eluted material showing the biological activity, c) applying the biologically active material from step b) to a column of aminoethyl cellulose equilibrated with an 0.01 molar aqueous buffer of pH 5.5 composed of volatile components, eluting the column with a buffer of the same molarity and then with a buffer having a concentration gradient of from 0.01 molar to 0.3 molar, detecting the presence of three peaks of eluted material each showing the biological activity and collecting separately the material of each peak which contains α- urogastrone, #-urogastrone or γ-urogastrone, respectively, followed by, in the case of material from the peak from step (c) containing #-urogastrone, applying the material to a column of carboxymethyl cellulose equilibrated with an 0.01 molar aqueous buffer of pH 5.0, eluting the column with a buffer of pH 5.0 with a concentration gradient of from 0.01 molar to 0.5 molar, collecting the eluted material showing the biological activity, applying this material to a column of a porous polyacrylamide gel excluding molecules of molecular weight greater than 5,000 in an 0.05 molar aqueous buffer of pH 7.2 and composed of volatile components, eluting the column with the same solution and collecting the eluted material showing the biological activity: or followed by in the case of material from the peak from step (c) containing γ-urogastrone, applying the material to a column of carboxymethyl cellulose equilibrated with an 0.01 molar aqueous buffer of pH 4.8, eluting the column with a buffer of pH 4.8 with a concentration gradient of from 0.01 molar to 0.5 molar, collecting the eluted material showing the biological activity applying this material to a column of a porous polyacrylamide gel excluding molecules of molecular weight greater than 5,000 and an 0.05 molar aqueous buffer of pH 7.2 and composed of volatile components, eluting the column with the same solution and collecting the eluted material showing the biological activity. The invention also comprises #-Urogastrone, being a white, water soluble, acidic polypeptide consisting of a single polypeptide chain with three internal disulphide bonds, having a molecular weight of about 6,000, showing the following physical properties:- paper chromatography in n-butanol: acetic acid: pyridine: water (30:6:24:20)-single spot R F  0.59; paper electrophoresis in pyridine acetate at pH 6.5-a small oval area moving anodically; paper electrophoresis in acetic acid/formic acid at pH 2.1-a 'comet' with front mobility of 1.75 relative to # -DNP lysine; acrylamide gel electrophoresis in 0.1M tris/hydrochloric acid buffer at pH 8.9-a single spot moving towards the anode with a mobility of 0.53 relative to bromophenol blue; isoelectric point-pH 4.5; giving an aminoacid ratio on analysis of aspartic acid 7, serine 3, glutamic acid 5, proline 1, glycine 4, alanine 2, valine 3, cysteine 6, methionine 1, isoleucine 2, leucine 5, tyrosine 5, histidine 2, lysine 2, tryptophan 2 and arginine 3, and having a specific biological activity, as defined earlier; of 0.2 to 0.5 ug/kg. and γ-Urogastrone, being a white, water soluble, acidic polypeptide consisting of a single polypeptide chain with three internal disulphide bonds and having a moleculan weight of about 6,000, showing the following physical properties: paper chromatography in n-butanol: acetic acid: pyridine: water (30: 6:24:20)-single spot R F  0.63; paper eleotrophoresis in pyridine acetate at pH 6.5-a small oval area moving slightly anodically; paper electrophoresis in acetic acid/formic acid at pH 2.1-a 'comet' with front mobility 1.57 relative to #-DNP lysine; acrylamide gel electrophoresis in 0.1M tris/hydrochloric acid buffer at pH 8.9-a single spot moving towards the anode with a mobility of 0.65 relative to bromophenol blue; isoelectric pointpH 4.3; giving a amino-acid ratio on analysis of aspartic acid 7, serine 3, glutamic acid 5, proline 1, glycine 4, alanine 2, valine 3, cysteine 6, methionine 1, isoleucine 2, leucine 5, tyrosine 5, histidine 2, lysine 2, tryptophan 2 and arginine 2, and having a specific biological activity, as defined earlier, of 0.2 to 0.5 ug/kg Pharmaceutical compositions for inhibiting gastric acid secretion are disclosed.