Structural studies on bovine prothrombin. Isolation and partial characterization of the Ca2+ binding and carbohydrate containing peptides of the N-terminus region.

Abstract

Three peptides, one of which binds Ca2-+ (calcium binding fragment, CBF) but contains no carbohydrates and two of which bind no Ca2-+ but contain carbohydrates, have been isolated from the N-terminus region of bovine prothrombin. The preparation of these peptides involved (a) thrombin cleavage of prothrombin to intermediate 1 (thrombinogenic) and fragment 1 (nonthrombinogenic), (b) tryptic attack on fragment 1, and (c) separation of the CBF from the latter reaction by addition of a phosphatidylcholine-phosphatidylserine dispersion in the presence of Ca-2+. Further study on the non-calcium-binding peptides from the tryptic digest of fragment 1 revealed the presence of two low molecular weight glycopeptides, GP-1 and GP-2. A detailed examination of the chemical characteristics of CBF provided some insight into this unusual peptide. Whereas fragment 1, as well as prothrombin, exhibited two classes of Ca-2+ binding sites (one of high affinity, 3-4 mol/mol of peptide and the other of low affinity, 10-12 mol/mol of peptide), CBF bound only 3-4 mol of Ca-2+/mol of peptide. This indicated the presence of only the high affinity sites of the parent molecule. CBF contained an unusually high level of glutamic acid (approximately 30% of the total amino acids as determined in an acid hydrolysate) and had an N-terminal glycine. Most likely these glutamyl residues were present originally as the gamma-carboxyglutamyl residue as proposed by Stenflo et al. (Stenflo, J., Ferlung, P., Egan, W., and Roepstorff, P. (1974), Proc. Natl. Acad. Sci. U.S-A 71, 2730). The CBF contained no detectable carbohydrate. Its molecular weight varied inexplicably according to the procedure used and gave the following values; 8500, by gel filtration; 5200, by 6 M guanidine-HCl gel chromatography; 3490, by analytical ultracentrifugation. The glycopeptides, GP-1 and GP-2, were distinguished from each other by differences in their behavior on ion exchange chromatography and in their amino acid composition, and from CBF by their inability to bind calcium under any conditions. On the other hand, GP-1 and GP-2 had nearly identical levels of carbohydrate, 45.1 and 48.0 wt %, and possessed essentially the same percent distribution of carbohydrates: sialic acid, 16.5 plus or minus 0.5; mannose, 10.3 plus or minus 0.4; glucosamine, 11.2 plus or minus 0.1; galactose, 7.9 plus or minus 0.3. Their molecular weights were as follows: GP-1, 70000, by gel filtration; 6500, by 6 M guanidine-HCl gel chromatography; 4600, by ultracentrifugation; GP-2, 6500 by gel filtration; 6900, by 6 M guanidine-HCl gel chromatography; 1960, by analytical ultracentifugation. Though there are some obvious variations depending on method, this could be attributable to a probable error in v measurement on these carbohydrate containing peptides. The significance of these findings as they relate to prothrombin to thrombin conversion is discussed.