Open AccessAn Improved Procedure for Automated Edman Degradation Used for Determination of the N ‐Terminal Amino Acid Sequence of Human Transcobalamin I and Human Intrinsic Factor
Author(s) -
THOMSEN Johannes,
BUCHER Ditlef,
BRUNFELDT Kay,
NEXØ Ebba,
OLESEN Henrik
Publication year - 1976
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1976.tb10861.x
Subject(s) - edman degradation , cleavage (geology) , serine , amino acid , intrinsic factor , phenyl isothiocyanate , chemistry , threonine , peptide sequence , biochemistry , vitamin b12 , biology , phosphorylation , paleontology , fracture (geology) , gene
An improved procedure for automated Edman degradation is presented. Three programs are described, one with double cleavage and two with single cleavage. The programs presented are characterized by a reversed delivery scheme for buffer, and phenyl isothiocyanate, and by reduced cleavage times. The modified procedures applied on automated Edman degradation of the vitamin B 12 ‐binding proteins human transcobalamin I and human intrinsic factor, containing approximately 390 and 350 amino residues respectively, gave the following N‐terminal amino acid sequences: Human transcobalamin I Glu‐Ile‐Cys‐Glu‐Val‐Ser‐Glu‐Glu‐Asn‐Tyr‐Ile‐Arg‐Leu‐Lys‐Pro‐Leu‐Leu‐Asn‐Thr‐Met‐Ile‐Gln‐Ser‐Asn‐Tyr‐Asn‐?‐Gly‐ Human intrinsic factor Ser‐Thr‐Gln‐Thr‐Gln‐Ser‐Ser‐Cys‐Ser‐Val‐Pro‐Ser‐Ala‐Gln‐Glu‐Pro‐Leu‐Val‐Asn‐Gly‐Ile‐Gln‐?‐Leu‐Met‐Glu‐Thr‐ The background accumulation seems to be related not only to the length of the polypeptide chain being degraded, but also to the content of serine (and possibly threonine). A possible N O acyl shift during the cleavage is a tentative explanation. The programs here represented lead to a significant reduction in background compared to conventional programs and allowed considerable prolongation of the degradations.