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B Hemmasi

Publications and source records attributed to B Hemmasi.

13 recordsLinked to original sources
PubMed1998

In vitro disulfide-coupled folding of guanylyl cyclase-activating peptide and its precursor protein.

Guanylyl cyclase-activating peptide II (GCAP-II), an endogenous ligand of particulate guanylyl cyclase C (GC-C), is processed from the precursor protein and circulates in human blood. GCAP-II consists of 24 amino acid residues and contains two disulfide bridges. The correct disulfide paring of GCAP-II is an absolute requirement for its biological activity. This study shows that the folding of the peptide from the reduced form yields a peptide with the native disulfide paring as a minor product and with non-native ones as major products, regardless of the presence or absence of reduced and oxidized glutathione. The results suggest that GCAP-II does not possess sufficient information to permit the adoption of the native conformation and to effectively form the correct disulfide pairing and, as a result, that GCAP-II is correctly folded by assistance of a factor(s) such as an intra- or intermolecular chaperone. We studied whether a peptide in the pro-leader sequence of the precursor protein (proGCAP-II) contains sufficient information to facilitate the folding of GCAP-II. For this purpose, we prepared proGCAP-II in Escherichia coli by a recombinant technique and examined the disulfide-coupled folding of proGCAP-II from the reduced form. proGCAP-II was quantitatively recovered with the correctly folded structure from the reduced form both in the presence and in the absence of reduced and oxidized glutathione. The protein contains only disulfide linkages at the same positions as the mature form of proGCAP-II, GCAP-II, and the biologically active isomer of GCAP-II in the molecule. These results provide evidence that the propeptide of proGCAP-II is a critical factor in the formation of the correct disulfide paring in the folding of the protein.

Amino Acid Sequence↗
PubMed1993

Synthesis of phytochelatins by the continuous flow solid phase procedure.

A nona- and an undecapeptide corresponding to phytochelatins with the general structure H-[gamma-Glu-Cys]n-Gly-OH were each synthesized by the continuous flow solid phase method using two different methodologies. Fmoc-amino acid derivatives were used as precursors, and two different H2N-POE-PS supports were employed. Different procedures were used to remove Acm protecting groups from Cys residues. In a second synthesis, Acm groups were removed before cleavage of the peptides from the polymer supports. The partially protected peptides of the first synthesis were purified by preparative HPLC. The purity and identity of all the synthesized peptides were verified by analytical HPLC and IS-MS and in some cases by amino acid analysis.

Amino Acids↗
PubMed1988

Continuous flow peptide synthesis on aminopolyoxyethylene-polystyrene graft copolymer using Fmoc-strategy.

An insoluble graft copolymer consisting of the covalently bound polyoxyethylene to cross-linked polystyrene (HO-POE-PS) was prepared by anionic polymerization of ethylene oxide on the resin. The copolymer was then converted to the corresponding amino-polymer (H2N-POE-PS) and the latter was employed as the solid carrier for peptide synthesis. Although HO-POE-PS has successfully been employed as a carrier for peptide synthesis by the standard shaking procedure using t-butoxycarbonyl-amino acids, now we deemed it of interest to test its suitability for the continuous flow synthesis. Thus, the C-terminal octapeptide of the porcine insulin B chain (B23-30) was prepared by this procedure using a photolabile anchoring group and fluoren-9-ylmethoxycarbonyl-amino acids. All the reactions were carried out in a continuous flow manner in a steel column under pressure using a high-performance liquid chromatography (HPLC) system. At the end of the synthesis, a sample of the protected peptide was cleaved from the support by photolysis. For the cleavage of another sample, an aqueous solution of sodium carbonate was employed. The protected peptide was purified on silica gel and Sephadex-LH 20. All the protecting groups of a sample of the octapeptide were removed with piperidine/dimethylformamide and trifluoroacetic acid and the deblocked peptide was purified by ion-exchange chromatography. The free peptide was shown to be homogeneous by thin-layer chromatography, HPLC, and electrophoresis. The identify of the free octapeptide was confirmed by amino-acid analysis, 13C-nuclear magnetic resonance measurement and field-desorption mass spectrometry. The peptide was also shown to be free of racemization.

Chromatography, High Pressure Liquid↗
PubMed1984

Synthesis of the C-terminal undeca- and protected docosapeptide of bovine insulin B-chain.

The synthesis of two C-terminal peptides of bovine insulin B-chain are described. Thus, insulin fragments (B9-30) and ( B20 -30) were synthesized using nitrobenzoylglycyl -poly-(oxyethylene) as the soluble support. 4-Carboxy-2-nitrobenzyl ester of Boc-alanine was coupled to glycyl-poly(oxyethylene) and the syntheses were continued employing symmetrical anhydrides of Boc-amino acids. The protected peptides were cleaved from the support by photolysis and were purified on silica gel and Sephadex LH-20. All the protecting groups of a sample of the undecapeptide were removed with liquid HF and the unprotected peptide was purified on CM-cellulose. The synthesized peptides were gas chromatographically tested for the racemization of the individual amino acids. The results indicated that no residue was significantly racemized .

Amino Acid Sequence↗
PubMed1983

Synthesis and photolytic cleavage of bovine insulin B22-30 on a nitrobenzoylglycyl-poly (ethylene glycol) support.

The synthesis of the C-terminal nonapeptide of bovine insulin B-chain is described. 4-(Bromomethyl)-3-nitrobenzoylglycyl-poly(ethylene glycol) Mr = 15,000) was used as soluble support. The C-terminal alanine was first converted to Boc-Ala-O-(2-nitro-4-carboxy) benzyl ester which was then coupled to Gly-PEG via DCC activation. The synthesis was performed using the in situ symmetrical anhydride coupling method. Cleavage of the protected peptide from the polymeric support was achieved by photolysis. The product was then chromatographed on a column of Sephadex LH-20. All the protecting groups of a sample were removed with liquid HF and the unprotected crude peptide was purified by ion-exchange chromatography on CM-Sephadex to obtain an electrophoretically and chromatographically pure peptide. The identity of this peptide was confirmed by field desorption mass spectrometry and amino acid analysis. Circular dichroism measurement suggests that the free nonapeptide possesses a disordered conformation. The nonapeptide was tested for the racemization of the individual amino acids by gas chromatography and the results showed that no residue was significantly racemized.

Amino Acid Sequence↗
PubMed1982

Syntheses of 4-[N-(tert-butoxycarbonyl)aminoacyloxymethyl]-3-nitrobenzoic acids for use in the liquid-phase peptide synthesis.

The syntheses of 4-(Boc-aminoacyloxymethyl)-3-nitrobenzoic acids are described. These compounds are suitable reagents for coupling to polyethylene glycol or its derivatives by the dicyclohexylcarbodiimide method to obtain 4-(Boc-aminoacyloxymethyl)-3-nitrobenzoyl-polyethylene glycol support. This is demonstrated by the reaction of the 4-carboxy-2-nitrobenzyl ester of Boc-alanine with glycylpolyethylene glycol in excellent yield. This synthetic route produces a much better coupling yield and an easier purification procedure in comparison with the original method in which Boc-amino acid cesium salts are allowed to react with 4-(bromomethyl)-3-nitrobenzoyl-polyethylene glycol.

Amino Acids↗
PubMed1979

Synthesis of the C-terminal decapeptide of bovine insulin B-chain.

The liquid-phase synthesis of a decapeptide corresponding to the last 10 amino acid residues of bovine insulin B-chain is described. Modified monofunctional polyethylene glycol containing benzyl bromide functional group was used as the soluble polymeric support. Cleavage of the fully-protected peptide from the polymer was achieved with 1N NaOH in dioxane. The protected peptide was purified by chromatography on Sephadex LH-20. The protecting groups of a sample were removed with anhydrous HF, and the unprotected crude decapeptide was purified by ion-exchange chromatography on carboxymethyl-cellulose. Both peptides were tested for the racemization of individual amino acids by the gas chromatographic method. The results showed that no residue had been significantly racemized.

Animals↗
PubMed1977

The solid phase synthesis of porcine secretin with full biological activity.

The solid phase synthesis of procine secretin is described. The C-terminal residue was attached to a polymeric amino support and all the Boc-amino acids, including Boc-glutamine, were coupled by a modified carbodiimide method. A preliminary test synthesis showed that the couplings of several amino acids of the N-terminal section were unsatisfactory. This problem was overcome in the main synthesis by executing all the major reactions twice. Cleavage of the peptide fromthe resin as well as the removal of all the side chain protecting groups was achieved with liquid HF. The product was purified by ion-exchange chromatography on SP-Sephadex to obtain a highly purified heptacosapeptide amide with full biological activity.

Amino Acid Sequence↗
PubMed1975

Ligand-exchange chromatography of amino acids on copper-, cobalt- and zinc-chelex 100.

Procedures for the ligand-exchange chromatography of amino acids on copper-, cobalt-and zinc-Chelex 100 have been examined. Ligand exchange on the copper complex affords a simple and rapid method for the removal of amino acids (except for aspartic and glutamic acids) from dilute solutions. The influence of the pH on the binding of amino acids to the metal complex was also studied. The bound amino acids could be eluted with ammonium hydroxide which also causes a slight metal leakage. Chromatography on cobalt- and zinc-Chelex 100 showed that only the basic amino acids were quantitatively attached to these complexes at pH 8.3-9.5, whereas the others were predominantly EXCLUDED. This procedure can be used for the selective concentration and removal of basic amino acids in the presence of other amino acids.

Amino Acids↗
PubMed1975

Ligand-exchange chromatography of amino acids on nickel-Chelex 100.

A ligand-exchange chromatographic proceudre for the selective separation of amino acids from inorganic ions is presented. It was found that the binding of amino acids to the nickel-Chelex 100 resin is pH dependent. At pH 8.5-9.1, only the basic amino acids lysine, histidine and arginine are quantitatively attached to the complex, whereas at pH 11, other amino acids with the exception of aspartic acid and glutamic acid are also bound, although not quantitatively. All of the amino acids can be eluted from the complex with 3 M ammonia solution without the displacement of nickel ions from the complex. This method can be used for the removal of the basic amino acids from solutions in the presence of inorganic ions as well as other amino acids.

Absorption↗