Add time:08/09/2019         Source:sciencedirect.com

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 α-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 α-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. ○ 1997 Elsevier Science Ltd

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