Synthesis of a hydrophilic affinity matrix for the purification of the vasoactive intestinal peptide receptor.
Vasoactive intestinal peptide (VIP) was assembled on a polyacrylamide gel using a combination of the Boc and Fmoc peptide synthesis strategies. Before the synthesis, the polymeric matrix functionalized with sarcosine methylester was treated with ethylenediamine in order to form primary amine reaction sites (0.3 mmol/g). Then a six-carbon spacer arm, Boc-aminocaproic acid, was coupled to the gel after activation with benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) reagent. After acidolysis of the Boc protecting group, the derivative Boc-asparaginyl (xanthenyl)-4-(oxymethyl)phenylacetic acid was introduced into the polyacrylamide resin. Leucine-27 and isoleucine-26 were incorporated into the peptide chain as Boc-protected derivatives while the subsequent amino acids were all introduced as Fmoc residues. All couplings were achieved with BOP reagent in presence of diisopropylethylamine. The synthesis proceeded easily and only asparagine-9 required a double coupling step. After completion of the VIP assemblage, the side-chain protecting groups were removed by reaction with trifluoroacetic acid containing appropriate scavengers. A sample of peptide-resin was treated with hydrofluoric acid and the quality of the synthetic VIP-COOH material, obtained after cleavage, was assessed by reverse-phase HPLC and fast atom bombardment mass spectrometry. The compatibility with aqueous solutions of the polyacrylamide resin loaded with VIP (0.15 mmol/g), as well as its ability to be utilized as an affinity matrix for VIP receptors, was demonstrated using solubilized receptor preparation made from porcine liver membrane. After a single-step affinity chromatography, no binding activity was anymore detectable in the pass-through fraction.(ABSTRACT TRUNCATED AT 250 WORDS)
Fournier
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Institut National de la Recherche Scientifique-Santé (INRS-Santé, Université du Québec, Pointe-Claire, Canada.
Couvineau
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Laburthe
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M
D000180:Acrylic Resins / Q000737:chemistry*; D000596:Amino Acids; D002846:Chromatography, Affinity / Q000379:methods; D005449:Fluorenes; D005563:Formic Acid Esters; D007202:Indicators and Reagents; D007532:Isoleucine; D007930:Leucine; D008099:Liver / Q000737:chemistry; D008566:Membranes / Q000737:chemistry; D009943:Organophosphorus Compounds; D011964:Receptors, Gastrointestinal Hormone / Q000302:isolation & purification*; D018005:Receptors, Vasoactive Intestinal Peptide; D014660:Vasoactive Intestinal Peptide / Q000737:chemistry*
D000180:Acrylic Resins; D000596:Amino Acids; D005449:Fluorenes; D005563:Formic Acid Esters; D007202:Indicators and Reagents; C016456:N(alpha)-fluorenylmethyloxycarbonylamino acids; D009943:Organophosphorus Compounds; D011964:Receptors, Gastrointestinal Hormone; D018005:Receptors, Vasoactive Intestinal Peptide; C016680:polyacrylamide gels; C000345:t-butyloxycarbonyl group; D007532:Isoleucine; D014660:Vasoactive Intestinal Peptide; C031032:benzotriazol-1-yloxy-tris(dimethylamino)phosphonium; D007930:Leucine