Determination of Iodoamino Acid Composition of Rat Thyroidal Iodoprotein: Some Sources of Serious Error1

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Abstract
Similar patterns of distribution of 131I in the iodoamino acids of labeled thyroid glands were found after hydrolysis by different enzymes and fractionation by paper chromatography in different solvent systems; the results suggest that these techniques may reveal the labeling pattern in intact thyroid glands, but only when certain precautions are taken in the experimental procedures employed. DIT consistently accounts for a larger share of thyroidal label, and chromatographic origin material for a smaller share, in Pronase hydrolysates than in those of Viokase, suggesting that the origin material of Viokase hydrolysates is richer in DIT than in the other thyroidal iodoamino acids. A severe deficiency of labeled thyroxine and triiodothyronine exists in centrifuged supernatants of thyroid hydrolysates (centrifuged after hydrolysis) compared to “whole” hydrolysates, probably due to preferential adsorption of the iodothyronines to the particulate matter of the hydrolysates. Because of tins phenomenon there is also a considerable apparent loss of iodothyronines even in “whole” hydrolysates when aggregation of the particulate matter occurs, as it does, frequently, when the hydrolysis period is prolonged or when freezing and thawing of the hydrolysates intervenes between the end of the hydrolysis period and chromatography. It is suggested that differences in the handling of thyroid hydrolysates are in part responsible for the discrepancies that are apparent when data pertaining to thyroidal distribution of radioiodine obtained by various investigators are compared. (Endocrinology75: 776, 1964)