Factors Influencing the Interaction Between Radioactive Iodine and Fibrinogen

Radioactive iodine is currently used to label proteins as a method of investigating protein metabolism in vivo or as a tracer for studies in vitro. However, recent observations indicate that changes in the molecular characteristics of proteins after iodination may lead to an apparent increase in the turnover rate in vivo (Regoeczi, 1971 ; Krohn et al., 1972). Incorporation of iodine into proteins has also been used to determine certain characteristics of protein structures (Cohen, 1968). We report here the effect of increased iodination on the properties of fibrinogen in vitro and also the changes in the extent of iodination of subunits of fibrinogen under various conditions. Human fibrinogen obtained from Abbott Universal Ltd. was used in all experiments. Fibrinogen was iodinated with 1251 by the iodine monochloride method of McFarlane (1963) substituting 0.5, 1.0,2.0,4.0,8.0 and 16 atoms of iodine/molecule of fibrinogen. lZ5I was added to each preparation as a radioactive marker and at the end of the reaction free iodide was removed by dialysis. Fibrinogen samples were subsequently dissolved in a mixture containing 4M-Urea, 2~-sodium dodecyl sulphate, 0.2~~2-mercaptoethanol and the subunits were separated by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis. The gels were stained and the radioactivity associated with the separated a, /3 and y chains was determined in a Packard Autogamma counter. After iodination at pH7.5, resulting in incorporation of 0.5 atoms of iodine/molecule of fibrinogen, the relative radioactivity associated with the subunits was 2.6 for the a chain and 3.3 for the B chain as compared with the radioactivity of the y chain which was used as the reference unit (1.0). With increasing degrees of iodination with lZ5I, the radioactivity of the y chain increased proportionately more than that of the a and /%chains reaching a ratio of 2.0 for the a chain, 2.1 for the B chain and 1.0 for the y chain under the conditions of iodination of 16 atoms of iodine/molecule of fibrinogen. The relative iodination of fibrinogen subunits was also a function of the pH of the iodinating mixture in the range pH5.5-10.0 studied. At 0.5 atoms of iodine/molecule of fibrinogen, the effect of pH on the relative lZ5I content of the subunits is shown in Table 1. Values are expressed as ratios of incorporation of radioactivity into a or jl chains of fibrinogen/radioactivity incorporated into y chains. The effect of iodination on the clotting time of the fibrinogen was determined by the addition of thrombin (0.25 N.I.H. units/mg of fibrinogen) to each preparation. The clotting time of the radioactive fibrinogen was calculated as a percentage of that of uniodinated fibrinogen and was found to decrease with increasing degrees of