Complex Between Cyanogen-Bromide-Activated
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Proc. Nat. Acad. Sci. USA Vol. 72, No. 3, pp. 1055-1058, March 1975 Structure of a Soluble Super-Active Insulin Is Revealed by the Nature of the Complex Between Cyanogen-Bromide-Activated Sepharose and Amines (N-substituted isoureas/N1-N2-disubstituted guanidines/insulin- Sepharose/insulin insensitivity/ super-active peptide hormones) MEIR WILCHEK*, TAKAMI OKA, AND YALE J. TOPPER National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014 Communicated by Bernhard Witkop, December 31, 1974 ABSTRACT Insulin-like material with elevated in- anhydride in 0.1 M NaHCO3. Crystalline porcine zinc insulin sulin specific activity is released from insulin-Sepharose was a gift from Eli Lilly Co. '251-Labeled porcine insulin was a in the presence of bovine-serum albumin. The mechanism of release and the chemical nature of this insulin-like gift from Dr. J. Roth. Sepharose-4B was purchased from material are revealed by the finding that amine- Sepharose Pharmacia Fine Chemicals. is O-Sepharose-N-substituted isourea. Nucleophilic attack Thin-layer chromatography on silica-gel, used for identifica- by amino groups releases N1-N2-disubstituted guanidines. tion of guanidine and guanidine derivatives, was performed Correspondingly, it is shown that the super-active insulin- in three different systems: (a) 1-butanol: acetic acid: H20, like material is an N1-N2-disubstituted guanidine in which insulin and bovine-serum albumin are the substituents. 4:1:5; (b) 1-propanol: NH40H, 7:3; (c) 1-butanol: pyridine: acetic acid: H20, 15:10:3:12. Compounds containing free amino groups can be covalently Sepharose-4B was activated with cyanogen bromide as bound to water-insoluble polysaccharides that have been described previously (1). The coupling of ammonia or N-a- reacted with cyanogen bromide at alkaline pH (1). This tech- acetyl ornithine to activated Sepharose was performed at pH nique has been widely used (2) for immobilization of biolog- 9 at 40 for 16 hr. The coupling of insulin to activated Sepha- ically active molecules such as enzymes, antibodies, and rose was performed as described previously (6, 7). The release hormones, and has also been used for the preparation of of bound ligands, other than insulin, from Sepharose was insoluble adsorbents for affinity chromatography. effected at 350 for 24 hr. Release of insulin-like material Despite the many applications of the method, the nature of (ILM) from insulin-Sepharose, in the presence of bovine- the bonds formed between the amino group and the carbo- serum albumin, was accomplished as described previously (6), hydrate has not been fully characterized. Axen and coworkers except when specified otherwise. (3) suggested the formation of three different structures, The accumulation of a-amino['4C]isobutyric acid by mouse N-substituted carbamates, N-substituted imidocarbonates, mammary gland explants (6) and the oxidation of [1-'4C]- and N-substituted isoureas. The last structure was also sug- glucose to "4CO2 by mouse diaphragm (15), were measured as gested by Svenson (4) on the basis of isoelectric focusing described previously. studies. The diverse applications of the method cited above, the recent introduction of hydrophobic chromatography (5), RESULTS and the finding that a soluble super-active form of insulin can Scheme I represents the chemical reactions discussed in this be prepared by treating insulin-Sepharose with bovine-serum section. It was shown recently (8) that alkyl amines retain albumin (BSA) (6), have made it important to determine the their charge when coupled to Sepharose. This suggested that exact chemical structure of the amine-Sepharose complex. the major products of the reaction are N-substituted isoureas In this paper, chemical evidence is given which shows that (compound 2). The formation of N-substituted isoureas was N-substituted isoureas are the major products of the reaction supported further by the finding of Oka and Topper (6), that between amines and cyanogen bromide-activated Sepharose. soluble insulin-like material can be released (reaction C) from Also, a structure for the soluble, super-active insulin that is insulin-Sepharose with medium containing bovine-serum generated by treatment of insulin-Sepharose with BSA is albumin, while no such material was solubilized in the absence proposed. of added protein. In order to confirm the formation of the N-substituted MATERIALS AND METHODS isourea structure, cyanogen bromide-activated Sepharose Poly(L-ornithine) and bovine-serum albumin were purchased (compound 1) was reacted with 0.1 M ammonium hydroxide from Miles Laboratories. N-a-acetyl ornithine and N-a-acetyl that had been brought to pH 9.5 by addition of HC1, at 40 for arginine were purchased from Fox Chemical Co. N-a-acetyl 3 hr to form ammonia-Sepharose. Treatment with ammonium citrulline was prepared by treating citrulline with acetic hydroxide was then continued for 24 hr at 35°. After filtration, the pH of the filtrate was 12.5. Thin-layer chromatography Abbreviations: ILM, insulin-like material; BSA, bovine-serum (Materials and Methods) of the filtrate in three solvent sys- albumin. tems revealed a spot which migrated with the same RF values * On leave from The Department of Biophysics, The Weizmann as guanidine, and which gave the same color as guanidine Institute of Science, Rehovot, Israel. when sprayed with the Sakaguchi reagent. The formation 1055 Downloaded by guest on October 2, 2021 1056 Biochemistry: Wilchek et aP.Proc. Nat. Acad. Sci. USA 72 (1975) Seph Seph Seph NH2 Seph -1 NH -OH O-C=-N - CNBr NH2R NH2R' OH 11 + R-NH-C-NHR' A I B C - OH ,-OH - i OH ACTIVATION COUPLING SCHEME I. Activation of Sepharose (Seph), coupling with amines to form Sepharose-bound isoureas and release of substituted gua- nidines. of guanidine indicates that ammonia-Sepharose is an 0-sub- filtrate with 6 N HCO, amino-acid analysis indicated the stituted isourea (compound 2; R = H). presence of 2 ,umol of arginine and 0.4,mol of ornithine per ml Additional evidence that ammonia-Sepharose is an isourea of N-a-acetyl-ornithine-Sepharose. The release (reaction C) of derives from a study of its reaction (reaction C) with N-a- arginine from N-a-acetyl-ornithine-Sepharose was also acetyl ornithine and poly(L-ornithine). One milliliter of am- effected with 0.1 M ammonium bicarbonate. monia-Sepharose was stirred with 50 mg of either compound The following experiments show that insulin-Sepharose at pH 8.5 for 24 hr at 35°. After hydrolysis of the filtrate with (compound 2; R = insulin) is also a substituted isourea, and 6 N HC1, amino-acid analysis indicated the presence of 5 usmol that the soluble super-active form of insulin which is released of arginine. Since the starting materials contained no detect- when insulin-Sepharose is treated with BSA (6) has a BSA- able arginine, these results also indicate that ammonia- substituent (compound 3; R = insulin; R' = BSA). Incuba- Sepharose is an 0-substituted isourea. tion of l25I-labeled insulin-Sepharose in the presence of BSA The product of the reaction between alkyl amines and cy- resulted in the release of about 8% of the insulin into the anogen bromide-activated Sepharose (reaction B) is also an medium, assuming all the released radioactivity to be protein- isourea, as shown by the following experiments. Five milli- associated. The filtrate was passed through a column of liters of N-a-acetyl-ornithine-Sepharose containing 10 ,umol of Sephadex G-50. Fig. 1A shows that 80% of the radioactivity ornithine per ml of Sepharose were incubated with 5.0 ml of migrated with BSA, while only a small amount of radioactivity 0.8 M ammonium hydroxide for 24 hr at 35°. After filtration, migrated as smaller molecular species. In a control experi- thin-layer chromatography of the filtrate (compare Mlaterials ment, with a mixture of BSA and insulin, the two proteins and Methods) revealed two spots: the major spot migrated migrated separately (Fig. 1B). These results indicate that with N-a-acetyl arginine; the minor spot migrated with N-a- treatment of insulin-Sepharose with BSA leads to the forma- acetyl citrulline, and probably reflects base-catalyzed hy- tion of a soluble molecule containing both BSA and insulin. drolysis of the substituted isourea (compound 2). No free Based upon the results with model compounds, presented N-a-acetyl ornithine was detected. After hydrolysis of the above, it is concluded that the BSA-insulin complex is an N1-N2-disubstituted guanidine (compound 3; R = insulin; R' = BSA). It has been shown previously that the insulin-like material E (ILM) released from insulin-Sepharose in the presence of 0 E Oa BSA has super-insulin activity (6). In order to determine CM whether the BSA-insulin complex, isolated from the column, 0 a) 0 C- is responsible for the super-insulin activity of ILM, the fol- 0 lowing experiments were undertaken: ILM was generated by co D incubating insulin-Sepharose in sterile 2.5% BSA/Medium cc 199 for 24 hr at 370 (6), and was then placed on a Sephadex I G-50 column that had been equilibrated with Medium 199. I- During elution with Medium 199 the BSA could be visualized as a narrow red band, since the phenol red in the medium was in Medium Fraction Number largely bound to this protein. This band migrated in 0.1 M FIG. 1. (A) Gel filtration of the* BSA-insulin complex. One 199 as the labeled BSA-insulin complex migrated milliliter of 125I-labeled insulin-Sepharose (containing 500 ,Ag of NaHCO3 (compare Fig. 1A), and was immediately assayed red protein, and 1.25 X 105 cpm) was incubated at 350 for 36 hr with for its biological activity. Since large amounts of phenol 1.0 ml of a 5% solution of BSA that had been brought to pH 7.5 are cytotoxic, small fractions of the red band were assayed.