The Quantitation of Isotope Loss with Tyrosine Loss in the Canine Fibrinogen-Fibrin Transformation

Abstract

Summary

Fibrinogen from the plasma of mongrel dogs was isolated and purified by fractional precipitation with ammonium sulfate. Cold-insoluble material and ammonium sulfate were removed by dialysis at 4° against 0.005 M sodium citrate. Portions of the resulting highly purified fibrinogen (95.1% clottability by 280 mμ analysis) were labeled with ¹³¹I by the iodine monochloride method. The percentage of the protein-bound radioactivity which (i) remained in association with the collected fibrin after thrombin addition; and (ii) was released in association with the split-off peptides into the clot supernatant fluid was determined by utilizing two different clotting procedures. The clotting procedure commonly used in an isotope dilution technique for fibrinogen quantitation has shown that 85% of the radioactivity remains associated with fibrin and the remaining 15.5% is recoverable in the supernatant fluid. The utilization of a clotting procedure for the colorimetric determination of fibrinogen in which the color is a measure of the amino acid tyrosine has revealed that 85.1% of the radioactivity can be recovered in fibrin with 17.3% being released into the supernatant fluid. These results suggest that approximately 16% of the radioactivity of labeled canine fibrinogen is released in the fibrinogen-fibrin transformation. The simultaneous loss of tyrosine from the fibrinogen molecule in the formation of fibrin has been found to be 10.6% and thus of the 16% total isotope loss at least this amount can be accounted for as iodotyrosine. The remaining 4-5% is at present still unaccounted for, but may be in association with the histidine residues present in the split-off peptides.