Antithrombin-III Denver, a reactive site variant.
Antithrombin-III Denver is a mutant protein which differs from the normal in being defective in serine protease binding (Sambrano, J. E., Jacobson, L. J., Reeve, E. B., Manco-Johnson, M. J., and Hathaway, W. E. (1986) J. Clin. Invest. 77, 887-893). It was isolated from the blood of an individual heterozygous for the abnormal gene by: affinity separation on heparin-Sepharose to obtain an antithrombin fraction, and gel filtration of the species present following complexing of the antithrombin fraction with a small excess of thrombin. The reduced, S-carboxymethylated protein formed a mixture of soluble tryptic peptides which was fractionated on Vydac C18. A single, unique peptide not present in a parallel experiment with normal antithrombin-III was isolated. This peptide was identified by sequence analysis and synthesis to correspond to residues 394-399 in the known sequence of the inhibitor, with leucine replacing reactive site P'1 residue Ser394. Although chromatograms of the tryptic peptides from the normal and mutant proteins were otherwise indistinguishable, the existence of additional residue replacements is not excluded. Measurements of the rate of thrombin binding by the mutant protein with p-aminobenzamidine as a fluorescent indicator showed that the second-order rate constant is reduced drastically. Meaningful measurements with the mutant protein could only be made in the presence of heparin and revealed a reduction of about 4000-fold in the rate constant.
D000595:Amino Acid Sequence; D000990:Antithrombin III / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001665:Binding Sites; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D014644:Genetic Variation; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments; D013917:Thrombin; D014357:Trypsin
D010446:Peptide Fragments; C050481:antithrombin III Denver; D000990:Antithrombin III; D014357:Trypsin; D013917:Thrombin