Urea Denaturation of Antihemophilic (AHF) and von Willebrand (vWF) Factors in the Presence and Absence of Selected Amino Acids

AHF and vWF are functionally independent activities in human plasma and a large body of data suggests that they may be different proteins, including a striking difference in the rate and extent of denaturation by urea. Thus, concentrations of urea below 1.5M resulted in 100% loss of vWF, 70% loss of AHF and 35% loss of Factor VIII related antigen (VIII Ag) in two hours, while 3M urea caused a loss of 100% vWF, 90% AHF and 45% VIII Ag in 30 hours. Full activity of urea denatured vWF and VIII Ag could be restored by dialysis of the urea while the AHF activity returned to about 75% of the initial value. Substitution of acetamide for urea resulted in only partial denaturation of the AHF (50% of that obtained with urea) suggesting that urea may interact with these proteins through the formation of bifunctional hydrogen bonds. Lysine and structural analogs of lysine with free amino groups prevented urea denaturation of the AHF but urea denaturation of vWF was not prevented by these amino acids. These data suggest that loci i essential for the biological activity of AHF, vWF and VIII Ag are denatured by direct interaction with urea. Since VIII Ag and AHF are more resistant and slower than vWF in their reaction with urea, other bonds or groups are also important. The differences in denaturation, renaturation and protection against urea denaturation of AHF and vWF by selected amino acids could be explained if the reactive sites for each activity were located on different molecules as suggested by other in vitro and in vivo observations or were remote from one another on one molecule.