Carboxylic acid-modified polyethylene: a novel support for the covalent immobilization of polypeptides for C-terminal sequencing.
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 thorough the alpha-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 epsilon-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 degrees 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, 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).
Shenoy
N R
NR
Beckman Research Institute of the City of Hope, Division of Immunology, Duarte, California 91010.
Bailey
J M
JM
Shively
J E
JE
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002234:Carbodiimides / Q000737:chemistry; D004024:Dicyclohexylcarbodiimide / Q000737:chemistry; D004743:Enkephalin, Leucine / Q000737:chemistry; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry; D007093:Imidazoles / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011095:Polyethylenes / Q000737:chemistry*; D017421:Sequence Analysis / Q000379:methods*
D000596:Amino Acids; D002234:Carbodiimides; D007093:Imidazoles; D010455:Peptides; D011095:Polyethylenes; D004024:Dicyclohexylcarbodiimide; D004743:Enkephalin, Leucine; C006900:N,N-carbonyldiimidazole; C081611:1,3-diisopropylcarbodiimide; D005022:Ethyldimethylaminopropyl Carbodiimide