Metal ion-binding properties of avian thymic hormone

Lanthanide ion luminescence studies and ⁴⁵Ca²⁺-binding measurements were used to study the metal ion-binding properties of avian thymic hormone. The procedure used to isolate the protein-involving heat-treatment at 80°C, trichloroacetic acid precipitation, DEAE-agarose chromatography, and gel filtration-affords material that is deemed homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as well as the absence of a detectable tryptophan signal in the fluorescence emission spectrum. Avian thymic hormone exhibits a pI = 4.35 when subjected to isoelectric focusing through polyacrylamide gels. The two ion-binding sites are indistinguishable in their interactions with Ca²⁺ and Mg²⁺, displaying K_Ca = 8 nM and K_Mg = 68 μM. The Eu^{3+ 7}F₀ → ⁵D₀ excitation spectrum at pH 6 displays a peak at 5795.4 Å, with a shoulder at 5792.8 Å and is replaced at higher pH values by a broader spectrum with a maximum at 5784.8 Å and a shoulder at 5777.1 Å. The pK_a governing this spectral interconversion is 8.21. All of these properties are very similar to those observed with other parvalbumins. However, polyclonal antibodies to avian thymic hormone do not cross-react with the parvalbumin from chicken leg muscle, as judged by Western blot analysis-further evidence that avian thymic hormone and the muscle-associated chicken parvalbumin are indeed distinct proteins.