Some Biochemical and Electrophoretic Studies on Purified Prothrombin , Factor

S TUDIES of the chemical nature of prothrombin have indicated a glycoprotein structure.2 Data obtained on purified bovine prothrombin have shown the reducing sugar content to be approximately 5 per cent, based on a reference standard of galactose and mannose.35 Laki et al.5 reported a value of 6.5 per cent in terms of glucose, and 1.6 to 1.7 per cent hexosamine. Alexander6 and colleagues observed a value of 6.5 per cent reducing sugar in terms of glucose in a highly purified human prothrombin preparation. A considerable fraction of the carbohydrate is said to be freed during the activation of prothrombin by citrate, becoming soluble in trichloracetic acid,3 and 1iller and SeegersT have isolated a polysaccharide of glucose from this moiety. While there thus can be little doubt as to the glycoprotein nature of prothrombin, the carbohydrate composition described above may be considerably influenced by sugar-containing contaminants. In or(ler to obtain further insight into the structure and function of the carbohydrate of bovine prothrombin, its carbohydrate composition was reexamined in purified preparations from some of which other known clotting factors were effectively separated. Moreover, in addition to reducing sugar and hexosamine, the acid carbohydrate, neuraminic acid ( sialic acid ) #{176} was assayed and found to comprise the largest constituent of the total carbohydrate in bovine prothrombin preparations. More important, it was demonstrated that this carbohydrate is not essential to the prothrombin molecule for its ability to yield thrombin. Biochemical studies were also carried out on factors VII and X. These substances are frequently concentrated to a large extent in the prothrombincontaining glycoprotein fractions obtained by older fractionation technics” and have been (!ifficult to obtain prothrombin-free. More recently this has been achieved by means of starch gel electrophoresis.10” The isolated factors thus obtained were then studied for their protein and carbohydrate composition. Additional insight into the chemical nature of prothrombin, factor VII and