SYNTHESIS AND CHARACTERIZATION OF A RECOMBINANT HIRUDIN-ALBUMIN COMPLEX

The purpose of this investigation was to covalently bind recombinant h irudin (rHir) to albumin and compare alpha-thrombin inhibition by comp lexed rHir to rHir. rHir was radiolabelled with I-125 and covalently b ound to albumin using heterobifunctional cross-linking reagents. HPLC purification of the I-125-rHir-SMCC-albumin complex using gel filtrati on chromatography resulted in four elution peaks, with the main peak c ontaining an average M(r) of 78 kDa. This peak fraction also contained 63% (+/- 1.4%) of the total protein and 49% (+/- 6.8%) of the I-125-r Hir conjugated to albumin. Purification of unbound I-125-rHir from com plex was confirmed by SDS gel electrophoresis and autoradiography. I-1 25-rHir inhibition of alpha-thrombin, measured by an assay utilizing t he chromogenic tripeptide substrate H-D-Phe-Pip-Arg-pNA (S-2238), was observed to be non-competitive of linear mixed-type having a K-i of 1. 61 pM and and alpha K-i of 1.09 pM. In contrast, complexed I-125-rHir was found to be a pure, non-competitive inhibitor having a K-i of 15.6 pM showing a ten-fold increase. These results demonstrate that covale ntly bound I-125-rHir still maintains potent alpha-thrombin affinity w hile losing minimal inhibitory capacity. Thus, successful modification of I-125-rHir serves as the foundation for future alternative applica tions for this potent inhibitor.