Synthesis and characterization of a recombinant hirudin-albumin complex.
The purpose of this investigation was to covalently bind recombinant hirudin (rHir) to albumin and compare alpha-thrombin inhibition by complexed rHir to rHir. rHir was radiolabelled with 125I and covalently bound to albumin using heterobifunctional cross-linking reagents. HPLC purification of the 125I-rHir-SMCC-albumin complex using gel filtration chromatography resulted in four elution peaks, with the main peak containing 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 125I-rHir conjugated to albumin. Purification of unbound 125I-rHir from complex was confirmed by SDS gel electrophoresis and autoradiography. 125I-rHir inhibition of alpha-thrombin, measured by an assay utilizing the chromogenic tripeptide substrate H-D-Phe-Pip-Arg-pNA (S-2238), was observed to be non-competitive of linear mixed-type having a Ki of 1.61 pM and an alpha Ki of 1.09 pM. In contrast, complexed 125I-rHir was found to be a pure, non-competitive inhibitor having a Ki of 15.6 pM showing a ten-fold increase. These results demonstrate that covalently bound 125I-rHir still maintains potent alpha-thrombin affinity while losing minimal inhibitory capacity. Thus, successful modification of 125I-rHir serves as the foundation for future alternative applications for this potent inhibitor.
New England Deaconess Hospital/Harvard Medical School, Boston, MA 02215.
M D
MD
Phaneuf
R K
RK
Ito
F W
FW
LoGerfo
D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D006629:Hirudins / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism*; D013438:Sulfhydryl Compounds; D013917:Thrombin / Q000037:antagonists & inhibitors
D003432:Cross-Linking Reagents; D006629:Hirudins; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D012710:Serum Albumin, Bovine; D013917:Thrombin; C060225:desirudin