Effects of amidination and chemical cross-linking on human factor VIII (antihemophilic factor).
The bifunctional reagent dimethyl suberimidate, reacting with primary amino groups of proteins, was used to cross-link highly purified human factor VIII. Reaction products were reduced with beta-mercaptoethanol or treated with Rhizopus arrhizus triglyceride lipase. The proportions of the dissociated subunits and their oligomers were calculated from the relative staining intensities of individual bands following polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate. Low concentrations of dimethyl suberimidate (up to 0.5 mM) produced covalently linked dimers which retained full functional (coagulant and von Willebrand factor) activities. Treatment with increasing concentrations of dimethyl suberimidate resulted in an almost simultaneous appearance of both trimeric and tetrameric species, suggesting the existence of specific intra-dimer contacts. A parallel decrease of functional activities was observed at higher concentrations of dimethyl suberimidate. A monofunctional reagent (ethyl acetimidate), reacting similarly with primary amino groups, amidinated factor VIII at rates similar to dimethyl suberimidate. Up to 40% amidinated factor VIII retained full biological activities. We conclude that the most reactive lysine residues are not involved in the active sites responsible for either coagulant or von Willebrand activity.
D001777:Blood Coagulation; D055598:Chemical Phenomena; D002621:Chemistry; D004120:Dimethyl Suberimidate; D004492:Edetic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D005169:Factor VIII / Q000378:metabolism; D006801:Humans; D007094:Imides; D008049:Lipase / Q000378:metabolism; D008239:Lysine; D008623:Mercaptoethanol; D008970:Molecular Weight; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014841:von Willebrand Factor / Q000502:physiology
D007094:Imides; D014841:von Willebrand Factor; D004120:Dimethyl Suberimidate; D008623:Mercaptoethanol; D005169:Factor VIII; D004492:Edetic Acid; D008049:Lipase; D008239:Lysine