Chromatographic isolation and identification of methylated derivatives obtained from modified fibrinogen

Polymerization was accomplished in the fibrinogen system by methylation with diazomethane, thionyl chloride and dimethyl sulphate. The duration and extent of polymerization were dependent on the modifying agent which was applied to the fibrinogen system. When fibrinogen was methylated with a very narrow range group-specific methylating agent, like dimethyl sulphate, the polymerization process was accelerated and proceeded with a reduction in the extent of modification of that obtained with the other methylating reagents utilized in these experiments. Chromatographic analysis revealed that diazomethane and thionyl chloride induced both O-methylation and N-methylation, as well as esterification of the carboxylate groups of aspartic and glutamic acid in fibrinogen. However, dimethyl sulphate resulted primarily in esterification accompanied by a small amount of histidine methylation. The proposed mechanism for the polymerization is through the esterification of the aspartic and glutamic acid residues which produces an increased protein-protein interaction resulting in polymer formation.