SPECTROSCOPIC CHARACTERIZATION OF TICK ANTICOAGULANT PEPTIDE

Tick anticoagulant peptide (TAP) is a disulfide rich potent inhibitor of factor Xa. Although this peptide is of potential clinical utility, very little is known about its higher order structure. Therefore, the secondary structure of recombinant TAP (rTAP) has been examined by cir cular dichroism (CD) and Fourier transform infrared (FTIR) spectroscop y. Both techniques suggest that rTAP is rich in beta-sheet structure. Disulfide bonds play a significant role in the folding and structural stability of rTAP. This is apparent from the resistance of rTAP to flu orescence-detected unfolding by guanidinium chloride (Gdn-HCl), unless disulfides are first reduced. The protein's tryptophan and tyrosine r esidues exhibit greater solvent exposure upon reduction of the cystine s as indicated by fluorescence spectra and second derivative UV spectr oscopy. A considerable amount of beta-structure appears to be retained after reduction of disulfides, although the CD spectrum manifests an increased amount of disordered structure in the reduced peptide. While rTAP does not bind the hydrophobic fluorescence probe 2-p-toludinylna phthalene-6-sulfonate (TNS) at neutral or acidic pH, the reduced pepti de binds TNS at pH 2.0 but not at pH 7.0. The secondary structure of t he reduced peptide at pH 2 is, however, similar to that at pH 7 as jud ged by CD spectroscopy. The reduced form of rTAP at acidic pH thus res embles a molten globule-like state.