J. Biol. Chem., Vol. 266, Issue 21, 13560-13563, Jul, 1991

Determination of disulfide bond pairs and stability in recombinant tick anticoagulant peptide

M Sardana, V Sardana, J Rodkey, T Wood, A Ng, GP Vlasuk and L Waxman
Department of Biological Chemistry, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.

Tick anticoagulant peptide (TAP) is a potent and selective inhibitor of blood coagulation factor Xa (Waxman, L., Smith, D.E., Arcuri, K.E., and Vlasuk, G.P. (1990) Science 248, 593-596). The 60-amino acid sequence of TAP shows limited homology to Kunitz-type inhibitors, including cysteines at positions 5, 15, 33, 39, 55, and 59. For detailed biochemical and pharmacological studies, a recombinant version of TAP (rTAP) has been produced in yeast. To determine the arrangement of the disulfide bonds, rTAP was cleaved with trypsin and chymotrypsin and the purified peptides sequenced using a gas-phase sequenator. The positions of the disulfide bonds were assigned by identifying the cycle(s) at which di-phenylthiohydan-toin-cystine was released. The specific disulfide bridges, Cys-5 to Cys-59, Cys-15 to Cys-39, and Cys-33 to Cys- 55, are analogous to those in the prototype Kunitz-type inhibitor, bovine pancreatic trypsin inhibitor (BPTI). While treatment of BPTI with dithiothreitol rapidly and specifically reduced one disulfide bond, the reduction of disulfide bonds in rTAP proceeded at a slower rate and appeared to be nonspecific, reaching a maximum of two disulfides reduced. Reduced rTAP derivatized with either iodoacetic acid or iodoacetamide lost 59% of its inhibitory activity. In contrast, BPTI alkylated with iodoacetic acid inhibited trypsin half as well as the iodoacetamide derivative. Although the arrangement of disulfides in the two inhibitors is the same, their susceptibility to reduction is markedly different.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J.-Y. Chang Denatured States of Tick Anticoagulant Peptide. COMPOSITIONAL ANALYSIS OF UNFOLDED SCRAMBLED ISOMERS J. Biol. Chem., January 1, 1999; 274(1): 123 - 128. [Abstract] [Full Text] [PDF]

				J.-Y. Chang and J. Y. Chang A Two-Stage Mechanism for the Reductive Unfolding of Disulfide-containing Proteins J. Biol. Chem., January 3, 1997; 272(1): 69 - 75. [Abstract] [Full Text] [PDF]

				R. S. Schwartz, D. J. Holder, D. R. Holmes Jr, J. P. Veinot, A. R. Camrud, M. A. Jorgenson, and R. G. Johnson Jr Neointimal Thickening After Severe Coronary Artery Injury Is Limited by Short-term Administration of a Factor Xa Inhibitor : Results in a Porcine Model Circulation, April 15, 1996; 93(8): 1542 - 1548. [Abstract] [Full Text]