Structural differences in active site-labeled thrombin complexes with hirudin isoinhibitors.
Hirudin, a 65 amino acid polypeptide from the medicinal leech, is the most potent thrombin inhibitor known to date. Recently, recombinant forms have been reported, which are as effective as the isolated forms. The studies presented here demonstrate sensitive spectroscopic methods for distinguishing binding of two recombinant hirudins, HV1 and HV2-Lys 47, with active site-labeled human alpha-, epsilon- and zeta-thrombins. Specifically, fluorosulfonylphenyl nitroxide spin labels, dansyl fluoride and p-nitrophenylanthranilate, were employed as active site-directed covalent reporter groups. In general, the nitroxide immobilization was greater for spin-labeled thrombin complexes with HV2-Lys 47 vs. HV1. The two fluorophore moieties, dansyl and anthraniloyl, were also sensitive to differences in HV1 and HV2-Lys 47 binding, including interactions with loop 145-150 of the thrombin structure where the epsilon- and zeta-thrombin cleavages exist. Speculation over sequence differences between the two isoinhibitors centers on residues 24 and 47, both of which involve either a loss or gain of charge on the side chain.
Rowand
J K
JK
Department of Chemistry, Ohio State University, Columbus 43210.
Berliner
L J
LJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D003619:Dansyl Compounds; D004578:Electron Spin Resonance Spectroscopy; D005456:Fluorescent Dyes; D006629:Hirudins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009589:Nitrogen Oxides; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013113:Spin Labels; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism*; D062367:ortho-Aminobenzoates
D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D006629:Hirudins; D009589:Nitrogen Oxides; D011994:Recombinant Proteins; D013113:Spin Labels; D062367:ortho-Aminobenzoates; C016616:4-nitrophenyl anthranilate; C016338:dansyl fluoride; D013917:Thrombin