The Properties of Thyroglobulin VIII. THE IODINATION OF THYROGLOBULIN

It has been shown that when thyroglobulin is iodinated, a marked intensification occurs in its absorption spectrum in the region of 300 to 3fXl rnp in alkaline solution (1). It is also well known that the absorption peaks of iodinated tyrosine and thyronine derivatives occur in this spectral zone (2). A procedure based on these factors has been developed which permits the simultaneous determination of the concentrations of the tyrosine, monoiodotyrosine, diiodotyrosine and thyroxine residues in thyroglobulin by measuring the increase in absorbancy that occurs between pH 5 and 12.35 at four wave lengths. The necessity of hydrolyzing the protein and isolating the amino acids is thereby obviated. The spectral analysis is accomplished rapidly on a small amount of protein with routine instrumentation. The problem at hand is to measure simultaneously, by photometric means, the concentration of tyrosine, monoand diiodotyrosine, and thyroxine in native (and iodinated) thyroglobulin preparations. The spectra of these four amino acids overlap appreciably and occur against a background of tryptophan absorption, which is also present in thyroglobulin. The two principal factors that permit a spectroscopic approach to this problem are (a) the marked increase that occurs in the dissociation constant of the phenolic hydroxyl group each time an iodine atom is substituted into the benzene ring and (b) the shift toward longer wave lengths and the intensification in absorption that occur with iodination. The spectra of tyrosine, monoand diiodotyrosine, and thyroxine show absorption at increasingly longer wave lengths, respectively, when compared in either their ionized or un-ionized forms. Pertinent spectral and pK data appear in Table I. The analytical method has been used to study the sequence of tyrosine iodination and iodotyrosine coupling when thyroglobulin is iodinated in vitro. The effect of the primary, secondary and tertiary structure of the protein on this process has been investigated also by iodinating (a) the products of the proteolytic digestion of thyroglobulin and (a) thyroglobulin in 8 M urea.