title: Reevaluating the effects of tyrosine iodination of recombinant hirudin on its thrombin inhibition kinetics.
abstract: To reevaluate the effects of iodination of hirudin on its thrombin-inhibiting activity, we iodinated a recombinant hirudin analog, CX-397, by a chloramine-T method and isolated the eight kinds of iodinated derivatives by reverse-phase HPLC. Their structure were different combinations of non-, mono-, or di-iodinated residues at Tyr-3 and Tyr-64. Their ability to inhibit alpha-thrombin were determined and compared with each other. Iodination at Tyr-64 slightly increased the association rate constant (Kon) 1.26- to 1.79-fold; this may be explained by the increase in the negative charge at C-terminal region of CX-397 caused by the electrophilic ortho substitution of Tyr-64 with iodine. On the contrary, iodination at Tyr-3 caused dual effects on the interaction between CX-397 and alpha-thrombin; namely, mono-iodination at Tyr-3 caused a small decrease in Koff (1.29- to 1.63-fold) than non-iodinated ones, whilst di-iodination at Tyr-3 brought about 3.04- to 3.88-fold increase in Koff. Since the N-terminal region of hirudin has been reported to bind with thrombin by hydrophobic interaction, the destabilizing effect of di-iodination at Tyr-3 can be explained by the increase in its polar nature, whereas the opposite effect of mono-iodination can not. These results indicate that it is not appropriate to use a mixture of variably radioiodinated hirudins as a radiotracer. We also provide a simple method to obtain well characterized radioiodinated hirudins.
authors.affiliation: Pharmaceuticals & Biotechnology Laboratory, Japan Energy Corporation, Saitama, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Komatsu
authors.forename: H
authors.initials: H
authors.lastname: Hayashi
mesh_terms: D000595:Amino Acid Sequence; D000991:Antithrombins / Q000737:chemistry* / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D006629:Hirudins / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D007455:Iodine; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D013917:Thrombin / Q000037:antagonists & inhibitors*; D014443:Tyrosine / Q000737:chemistry
chemical_list: D000991:Antithrombins; D006629:Hirudins; D011994:Recombinant Proteins; D014443:Tyrosine; D007455:Iodine; D013917:Thrombin