Thyroxine-binding globulin: investigation of microheterogeneity

Preparations of T4-binding globulin (TBG) from human serum was performed using only two affinity chromatography steps. Purity of the protein was demonstrated by a single band in overloaded disc and sodium dodecyl sulfate electrophoresis, equimolar binding to T4, and linearity in sedimentation velocity run. The molecular weight was calculated to be 60,000 +/- 3,000 daltons (n = 3), the sedimentation coefficient was 3.95S, and the Stokes' radius was 37 A. The amino acid composition was found to be in good agreement with the calculations of other authors. By isoelectric focussing (IEF), pure TBG showed four main bands at pH 4.25, 4.35, 4.45, and 4.55 together with several fainter bands. The N- acetylneuraminic acid (NANA) content of the four TBG bands isolated by preparative IEF was found to decrease from 10.2 mol NANA/mol TBG in the band at pH 4.25 to 4.8 mol NANA/mol TBG in the band at pH 4.55. No significant difference in the affinity constants of the TBG bands to T4 was found. The affinity constants for TBG ranged from 3.1 x 10(9) to 7.2 x 10(9) M-1. Sequential kinetic desialylation of pure TBG resulted in a progressive tendency toward one major band at pH 6.0. In native sera, microheterogeneity of TBG was detected after IEF on polyacrylamide gel plates by immunofixation. The typical TBG patterns shown by pure TBG were also found in normal subjects. Characteristic deviations from this pattern were found in the sera of females during estrogen therapy or pregnancy, where there was a gradual increase in density of the band at pH 4.25 and the appearance of an additional band at pH 4.15. In sera from patients with liver disease and elevated TBG levels, there was a fading of the acidic bands, whereas the more alkaline band at pH 4.55 was intensified. It is therefore proposed that microheterogeneity of TBG is caused by differences in NANA content and that variations of TBG patterns in native sera may reflect altered TBG synthesis or degradation. A genetically related microheterogeneity of TBG could not be demonstrated after examination of 800 sera, including 2 families with quantitative TBG deficiency