Thrombin-binding affinities of different disulfide-bonded isomers of the fifth EGF-like domain of thrombomodulin.
The fifth EGF-like domain of thrombomodulin (TM), both with and without the amino acids that connect the fifth domain to the sixth domain, has been synthesized and refolded to form several different disulfide-bonded isomers. The domain without the connecting region formed three disulfide-bonded isomers upon refolding under redox conditions. Of these three isomers, the (1-2,3-4,5-6) bonded isomer was the best inhibitor of fibrinogen clotting and also of the thrombin-TM interaction that results in protein C activation, but all the isomers were inhibitors in both assays. The isomer containing an EGF-like disulfide-bonding pattern (1-3,2-4,5-6) was not found among the oxidation products. The domain with the connecting region amino acids (DIDE) at the C-terminus formed two isolable products upon refolding in redox buffer. These products had the same two disulfide-bonding patterns as the earliest and latest eluting isomers of the domain without the DIDE. In order to compare the thrombin-binding affinities of these isomers to the isomer with the EGF-like disulfide bonds, acetamidomethyl protection of the second and fourth cysteines was used to force the disulfide bonds into the EGF-like pattern. Thrombin-binding affinity, measured as inhibition of fibrinogen clotting and as inhibition of protein C activation correlated inversely with the number of crossed disulfide bonds. As was found for the domain without the connecting region, the isomer that was the best inhibitor of fibrinogen clotting and of protein C activation was the isomer with no crossing disulfide bonds (1-2,3-4,5-6).(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Chemistry and Biochemistry, University of California-San Diego, La Jolla 92093-0601, USA.
M J
MJ
Hunter
E A
EA
Komives
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides; D004815:Epidermal Growth Factor / Q000737:chemistry*; D007536:Isomerism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013917:Thrombin / Q000378:metabolism*; D018180:Thrombomodulin / Q000737:chemistry* / Q000378:metabolism*
D004220:Disulfides; D010446:Peptide Fragments; D018180:Thrombomodulin; D004815:Epidermal Growth Factor; D013917:Thrombin