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

• Published in: Protein science 1992-01, Vol.1 (1), p.58-67
• Main Authors: Shenoy, Narmada R., Bailey, Jerome M., Shively, John E.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acids - analysis; Carbodiimides - chemistry; carboxylic acid‐modified polyethylene; C‐terminal sequencing; Dicyclohexylcarbodiimide - chemistry; Enkephalin, Leucine - chemistry; Ethyldimethylaminopropyl Carbodiimide - chemistry; Imidazoles - chemistry; Molecular Sequence Data; Peptides - chemistry; Polyethylenes - chemistry; Sequence Analysis - methods
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1002/pro.5560010107

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), 1,1′‐carbonyldiimidazole (CDI), 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride (EDC), benzotriazol‐1‐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 α‐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 × 5‐mm pieces for use in our continuous flow reactor (CFR), which is also used for automated N‐terminal sequencing (Shively, J.E., Miller, P., & Ronk, M., 1987, Anal. Biochem. 163, 517–529). Automated C‐terminal sequencing on these supports is described in the companion paper (Bailey, J.M., Shenoy, N.R., Ronk, M., & Shively, J.E., 1992, Protein Sci. 1, 68–80).