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  • Title: Core domain of hirudin from the leech Hirudinaria manillensis: chemical synthesis, purification, and characterization of a Trp3 analog of fragment 1-47.
    Author: De Filippis V, Vindigni A, Altichieri L, Fontana A.
    Journal: Biochemistry; 1995 Jul 25; 34(29):9552-64. PubMed ID: 7626625.
    Abstract:
    Hirudin is a small (approximately 7 kDa) disulfide-cross-linked polypeptide known as the most potent and specific thrombin inhibitor. We have previously shown that the N-terminal proteolytic fragment 1-47 of hirudin HM2 from Hirudinaria manillensis maintains inhibitory action toward thrombin [Vindigni, A., et al. (1994) Eur. J. Biochem. 226, 323-333]. Here we report the solid-phase chemical synthesis of an analog of fragment 1-47 bearing a Tyr3-->Trp exchange (Y3W analog). The crude, reduced peptide was purified by reverse-phase HPLC and subjected to oxidative folding to the disulfide-cross-linked species. The folding process of the Y3W analog was slower than that of the natural fragment 1-47, but nevertheless still occurred almost quantitatively as the natural species. The overall final yield of the synthetic product was approximately 35%, and its identity and homogeneity was established by a number of analytical techniques, including electrospray mass spectometry. The unique alignment of the three disulfide bridges of the Y3W analog was established by peptide mapping as Cys6-Cys14, Cys16-Cys28, and Cys22-Cys37 and shown to be identical to that of the natural fragment. The results of far- and near-ultraviolet circular dichroism and fluorescence emission measurements provided evidence that the Y3W analog retains the structural features of the natural species. The thermodynamic quantities (delta GD, delta Hm, delta Sm, and delta Cp) characterizing the reversible and cooperative thermal unfolding processes of the Y3W analog (Tm = 60.5 degrees C) and the natural fragment species (Tm = 62.5 degrees C) were evaluated. Despite the relatively high Tm values, the stability of both fragment species at 37 degrees C was only approximately 10 kJ mol-1, well below the average 50 kJ mol-1 typical of single-domain globular proteins. The synthetic Y3W species was found to be approximately 5-fold more active (KI = 30 +/- 5 nM) than the natural fragment 1-47 (KI = 150 +/- 20 nM) in inhibiting thrombin. Of interest was that the difference in the free energies of binding to thrombin at 37 degrees C, delta delta Gb, between the Y3W analog and natural species (4.2 kJ mol-1) was that expected for the difference in hydrophobicity between the two polypeptides resulting from the Tyr-->Trp exchange. The results of this study indicate that solid-phase chemical synthesis represents a convenient and high-yield procedure to prepare analogs of the biologically active, N-terminal core domain of hirudin with improved functional properties.
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