Carboxylic acid‐modified polyethylene: A novel support for the covalent immobilization of polypeptides for C‐terminal sequencing

Abstract

We have developed a method for the covalent immobilization of peptides, for the purpose of C-terminal sequencing, to a novel solid support, carboxylic acid-modified polyethylene (PE-COOH) film. The peptides are attached by coupling the N-terminal amino group to the activated carboxyl groups of the film. Reagents for carboxyl group activation, including 1,3-dicyclohexylcarbodiimide (DCC), 1,l'-carbonyldiimidazole (CDI), l-ethyl-3-(3-dimethylaminopropy1)carbodiimide hydrochloride (EDC), benzotriazol-l-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and 1,3-diisopropylcarbodiimide (DICD) were compared. The best yields were obtained with DCC for a variety of tested peptides and averaged approximately 50%. The covalent attachment at pH 6.7 of peptides was shown to occur predominantly through the a-amino group for the peptide, SIGSLAK, which after attachment to the PE-COOH support permitted the C-terminal lysine residue to be sequenced in good yield, indicating that the €-amino group of lysine is not covalently attached. This support offers a number of advantages over other solid supports, such as silica and polyvinylidene difluoride, for C-terminal sequencing including (1) stability to base and the high temperatures (65 "C) employed for C-terminal sequencing, (2) wettability with both aqueous and organic solvents, (3) a high capacity (1.6 nmol/mm2) for covalent coupling of polypeptides, and (4) easy divisibility into 1 X 5-mm pieces for use in our continuous flow reactor (CFR), which is also used for automated N-terminal sequencing (Shively,

Crown ether-modified electrodes for the simultaneous stripping voltammetric determination of Cd(II), Pb(II) and Cu(II)

“…The carboxyl groups of the electrografted diazonium salt were activated by incubating the functionalized electrodes in a 26 mM EDC and 35 mM sulfo-NHS solution in 100 mM MES buffer (pH 4.5) for 1 h. In order to conjugate the carboxy-fucntionalized electrode with the carboxy-modified ligands, a lysine spacer was intercalated in between, by using its two amino functionalities to form amido bonds [22][23][24][25]. The surface activated groups reacted overnight with the α-amine group of the lysine at 4°C.…”

Automated carboxy‐terminal sequence analysis of peptides and proteins using diphenyl phosphoroisothiocyanatidate

“…It was found that treatment of the covalently coupled peptide with DPP-ITC in the presence of a molar excess of pyridine at 50 "C for 40 min, conditions simi-lar to those employed in the solution phase, formed peptidylthiohydantoin. Cleavage of the derivatized C-terminal amino acid was accomplished with dilute aqueous triethylamine as described above (Bailey & Shively, 1990;Shenoy et al, 1992). The presence of thiohydantoin leucine was assessed by comparison of retention time with standard thiohydantoin leucine on reverse-phase HPLC.…”

Immobilization of D‐ribulose‐1,5‐bisphosphate carboxylase/oxygenase: A step toward carbon dioxide fixation bioprocess

“…Immobilization of Rubisco was carried out on nylon membranes using 1,3-dicyclohexylcarbodiimide (DCC) as described for other enzymatic proteins in previous reports (Agarwal and Bhattacharya, 1999;Shenoy et al, 1992). Strips sized 1 × 5 × 0.05 cm with a marked and dented circular area of 0.8-cm diameter were used to immobilize the Rubisco.…”

“…Twenty units of Rubisco in a final reaction volume of 50 L was applied onto the membrane in the localized (dented) circular area. Immobilization was initiated by the addition of 20 L (5 mM) of 1,3dicyclohexylcarbodiimide in the reaction mixture as described elsewhere (Shenoy et al, 1992). Each nylon membrane was soaked with 40 L of 1 g/L RuBP for 10-15 minutes (before washing with buffer).…”