Stokes radius determination of radioiodinated polypeptide hormones by gel filtration

A simple technique for determination of the molecular (Stokes) radius of radioiodinated proteins was developed using the same column and chromatographic conditions employed in routine radioimmunoassay tracer purification. The calibration curve for five radioiodinated standard proteins presented a highly significant correlation (r = -0.996; P less than 0.001) and allowed precise molecular radius determination for labeled human growth hormone (hGH), luteotropin (hLH), follicle-stimulating hormone (hFSH), thyrotropin (hTSH), prolactin (hPRL), and corticotropin (hACTH), enabling detection of differences of the order of +/- 3%. The validity of the method was verified by determining the molecular radius of hGH in both "cold" (unlabeled standards and unknowns) and "hot" (radioiodinated standards and unknowns) systems. The technique can be applied in a very simple manner, requiring just one simple additional calibration run before Sephadex G-100 tracer purification. Furthermore, it can be applied to any protein, even when only extremely limited amounts are available. Since the standards and unknowns are labeled and chromatographed under identical conditions, potential common alterations of the molecule due to oxidation, iodine incorporation, tracer-carrier interactions, etc., are automatically corrected for.