Fibrinogen-Induced Polymerization Via the Process of Methylation

Summary

Fibrinogen is polymerized by a number of group specific reagents including diazomethane, thionyl chloride and di-methyl sulfate at pH 7.4. The relationship between the number of methyl groups incorporated into fibrinogen and the extent of polymerization was evaluated. With diazomethane and thionyl chloride as modifying agents, polymerization ensued in approximately IV2 hr with extensive modification of fibrinogen. On the other hand, m�thylation via di-methyl sulfate-induced polymer formation occurs in approximately 35 min with primarily carboxylic acid group esterification. The polymerized fibrinogen formed under these conditions exhibited properties that were closely similar with the physiological fibrin clot.

Amino group determinations revealed the m�thylation of amino acid residues other than the expected esterification of carboxylic acid groups. Diazomethane induced both N- methylation of lysine, as well as O-methylation of tyrosine, as estimated from spectrophotometric analysis. On the other hand, thionyl chloride modified only a small number of amino groups, and di-methyl sulfate modification resulted in no significant amounts of amino group methylation during the process of modification induced polymerization of fibrinogen.

The profile of the number of methoxyl groups incorporated into fibrinogen with time for diazomethane modification may reflect a conformational change in the protein due to a more nonspecific m�thylation. Both the reagent and the conditions of modification were found to be important in achieving a selective modification of fibrinogen.

A possible interpretation of these results is the esterification of carboxylic acid groups in the fibrinogen with reduction in the prevailing carboxylate ion negative repulsion, thereby achieving an increased protein-protein interaction with a resulting polymerization.

• References

• 1 Bayley T, Clements JA, Osbahr AJ. 1967; Pulmonary and circulatory effects of fibrinopeptides. Circ. Res 21: 469-186
• 2 Blombäck B, Blombäck M, Gröndahl NJ, Holmberg E. 1966; Structure of fibrinopeptides - its relation to enzyme specificity and phylogeny and classification of species. Arkiv. Kemi. 25: 411-418
• 3 Colman RW, Morris RE, Osbahr AJ. 1967; New vasoconstrictor bovine peptide-B released during blood coagulation. Nature 215: 292-297
• 4 Gladner JA, Murtaugh PA, Folk JE, Laki K. 1963; Nature of peptides released by thrombin. Ann. N. Y. Acad. Sci., USA 104: 47-52
• 5 Laki K. 1951; The polymerization of proteins: the action of thrombin on fibrinogen. Arch. Biochem. Biophys. 32: 317-321
• 6 Laki K, Gladner JA. 1964; Chemistry and physiology of the fibrinogen-fibrin transition. Physiol. Rev. 44: 127-138
• 7 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951; Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265-270
• 8 Moore S, Stein WH. 1954; A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. J. Biol. Chem. 211: 907-915
• 9 Niederl JB. 1942. Micro Methods of Quantitative Organic Analysis. 2. ed New York: J. Wiley and Sons, Inc; pp 257-262
• 10 Osbahr AJ, Gladner JA, Laki K. 1964; Studies on the physiological activity of the peptide released during the fibrinogen-fibrin conversion. Biochim. Biophys. Acta 86: 535-542
• 11 Osbahr AJ. 1974; The hemostatic effect of bovine peptide-B from fibrinogen. Thromb. Diath. Haemorr. 32: 149-157
• 12 Osbahr AJ, Colman RW. 1974; Structure-activity relationships of the peptides released from canine fibrinogen by thrombin. Biochim. Biophys. Acta 336: 273-282
• 13 Osbahr AJ. 1975; Structure-activity relationships of the vasopressor activity of canine peptide-A from fibrinogen. Biochim Biophys. Acta 386: 373-382
• 14 Osbahr AJ, Custodio R. 1975; Action of peptide-B from bovine fibrinogen on ATPase activity and superprecipitation of myosine B. Am. J. Physiol. 228: 488-495