Equilibrium Density Centrifugation of Thyroglobulin in RbCI

A method of evaluating the iodine distribution in thryroglobulin has been developed. It is based on the increase in density that occurs when iodine is incorporated into proteins. Resolution of iodinated species is obtained by equilibrium centrifugation in a 34.5% (w/w) RbCl density gradient (isopycnic centrifugation) in either the analytical or preparative ultracentrifuge. The equilibrium in 34.5% RbCl of a human thyroglobulin preparation which contained almost no iodine was studied. A molecular weight of 6.3 x 105 and a density of 1.314 ± 0.001 g per cm3 were determined for this preparation. The increase in density of iodinated thyroglobulin was proportional to the amount of iodine incorporated. A computer analysis has been developed to determine the iodine distribution. The theoretical curves fit the data with high precision. Preparative gradient equilibrium centrifugation of radiolabeled thyroglobulin in 34.5% RbCl was also evaluated. Radioiodine was incorporated into thyroglobulin which was less dense than the unlabeled molecules. The density of newly synthesized l-[U-14C]valine thyroglobulin was less dense than that of radioiodine-labeled protein. It appears that synthesis and iodination of thyroglobulin are controlled by different metabolic processes.