Reduction of disulfides in urokinase and insertion of a synthetic peptide.
Abbokinase is a commercially available two-chain lower molecular weight urinary plasminogen activator (u-PA) with low affinity for fibrin. Therefore, therapeutic use of this u-PA form (UK) may cause activation of not only clot-bound plasminogen but also systemic plasminogen. Since this form of UK contains a 13-residue peptide remnant of the A chain disulfide-linked to the catalytic B chain, disulfide-exchange could allow replacement of this native peptide with peptides of desired clot-directed properties. We report here a method in which native peptide was partially removed by subjecting UK to 1-100 mM dithiothreitol (DTT), in the presence of arginine. Arginine (250 mM) was found to slow the rate of release of the peptide by 2-fold, to slow the loss in enzymatic activity by about 9-fold and to limit the extent of disulfide reduction. Upon dialysis to remove DTT from the reduced UK mixture, the disulfides reformed and enzymatic activity was regained. Synthetic peptide added at this point became incorporated to the level of about 0.4 mol/mol UK. This method should be very useful for developing UK derivatives with altered affinities toward fibrin clots.
Homandberg
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Abbott Laboratories, Abbott Park, IL.
Wai
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D000595:Amino Acid Sequence; D001120:Arginine; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides; D004229:Dithiothreitol; D005337:Fibrin / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000138:chemical synthesis / Q000378:metabolism
D004220:Disulfides; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D005337:Fibrin; D001120:Arginine; D014568:Urokinase-Type Plasminogen Activator; D004229:Dithiothreitol