Polyacrylamide Electrophoretic Study of Thyroxine Binding to Human Serum

Abstract

Polyacrylamide electrophoresis of normal human serum labeled with I131 T4 results in the separation of 4 distinct zones of radioactivity. Two of these zones migrate further than albumin, one is associated with albumin and one migrates less than albumin. The 4 areas of T4 radioactivity were arbitrarily named Peaks I, II, III, and IV. Ditnitrophenol (DNP) decreased the radioactivity associated with Peak I, barbital decreased the radioactivity in Peaks I and II, and diphenyl hydantoin reduced the radioactivity in Peak IV. From these observations and other evidence cited below, it was concluded that Peak IV in the present system was thyroxine binding globulin (TBG) as defined by paper electrophoresis; that Peak III represented T4 bound to albumin; that Peak II corresponds to thyroxine-binding prealbumin (TBPA); and that Peak I is free T4. The T4 capacity of Peak IV was 9.10+3. 45/ f g T 4/100 ml. No binding maxima for T4 in Peaks I, II and III were demonstrated. Rabbit antisera to the protein in Peaks HI and IV were produced. Immunoelectrophoresis using these antisera showed Peak IV to be associated with [alpha]-/globulin and Peak HI with albumin. Electrophoresis using paper, starch gel and polyacrylamide gel yields qualitatively or quantitatively different estimates of T4 binding by human serum, the in vivo translation of such data must be made cautiously.