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Characterization of Digoxigenin Derivatives of Human Coagulation Factors VIIa and X by Electrospray Mass Spectrometry and Enzyme Kinetics
Author(s) -
Thiruvikraman Singanallore V.,
Roboz John,
Wang Tsonghui,
Ma Longhua,
Seaman Glen,
Guha Arabinda
Publication year - 1996
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/(sici)1097-0231(199608)10:11<1367::aid-rcm653>3.0.co;2-p
Subject(s) - chemistry , dig , derivatization , enzyme , digoxigenin , electrospray ionization , mass spectrometry , chromatography , histidine , arginine , stereochemistry , biochemistry , amino acid , microbiology and biotechnology , in situ hybridization , gene expression , gene , biology
Digoxigenin ester (Dig) derivatives of coagulation factors VIIa and X facilitate staining studies to localize tissue factor activity in human atherosclerotic plaques. The larger the number of attached Dig units the easier it is to form highly visual stains. Electrospray ionization was used to characterize the Dig derivatives, as a function of excess derivatization reagent, to determine the optimal derivatives, i.e. the highest number of Dig units attached with the product still retaining enzymatic activity. The enzymatic activity of factor VIIa derivatized at 50‐fold Dig excess (with 28, 34, 48 or 52 Dig units attached, masses to 79 kDa) remained the same as that of native factor VIIa. In contrast, the enzymatic activity of factor X derivatives diminished above 15‐fold Dig excess (15 Dig units attached, mass 65.6 kDa). The distribution of derivatized lysine, histidine, arginine, tryptophan and tyrosine residues was estimated for several possible configurations.