Evaluation of the homogeneity of several thyroglobulin preparations.

A technique for the preparation of thyroglobulin by fractional precipitation with either ammonium sulfate or potassium phosphate buffer was described in 1948 by Derrien et al. (1). These preparations were found to be homogeneous by boundary electrophoresis (l), but heterogeneous in their salting-out curves (1) and in the ultracentrifuge (2, 3). In the ultracentrifuge, a main component with a sedimentation coefficient of 19 S was seen, as well as small amounts of both faster and slower sedimenting components. The heterogeneous appearance of these thyroglobulin preparations could be due to the presence of contaminating proteins. However, since it has been demonstrated that the thyroglobulin molecule will undergo dissociation under certain conditions of pH and ionic strength (3, 4, 5), it is also possible that the heterogeneity is due to dissociation and aggregation phenomena. The present study was undertaken to evaluate whether this heterogeneity is physical or chemical in nature, as well as to explore other procedures for the preparation of thyroglobulin. The homogeneity of the various preparations obtained was evaluated by starch gel electrophoresis and by ultracentrifugation, since it was found that heterogeneity was detected only by these techniques, and not by boundary electrophoresis, paper electrophoresis, or starch column electrophoresis. It was possible to demonstrate that the components seen by starch gel eleetrophoresis correspond to those seen in the ultracentrifuge. The preparative procedures studied included, in addition to phosphate fractionation, alcohol fractionation, DEAE-cellulose chromatography, ultracentrifugation techniques, and elution from starch gels after electrophoresis. The information obtained from these studies indicated that the heterogeneity present in thyroglobulin preparations is physical in nature and based on differences in molecular size. It is likely that dissociation and aggregation of the main thyroglobulin component are responsible for this heterogeneity and that these processes are accelerated by dialysis against distilled water and lyophilization.