Biological Activity of a Fluorescein Human Growth Hormone Derivative Prepared by Specific Covalent Labeling of Lysine-70

Modification of human growth hormone (hGH) with a low equimolar concentration of fluorescein isothiocyanate (FITC) yielded a derivative containing 1 mol of fluorescein/mol of protein. The site of modification was identified as lysine-70. Lysine-70 of hGH is about 3-fold more reactive than a “normal” lysine in a protein, having pseudo-first-order kinetics K_obs = 110 ± 7 M⁻¹ min⁻¹ at pH 10.5. The pK_a of the lysine was estimated to be 10.7, within the normal range of normal ε-lysine moieties in proteins. This higher chemical reactivity seems to favor selective labeling of this moiety at low FITC concentrations. To obtain monomodified derivatives, hGH was derivatized with 0.6 equiv of FITC, and the modified derivatives were separated from unreacted hormone by means of HPLC using a Mono Q column. Its biological activity, determined by Nb₂ bioassay, decreased to 40%, and its affinity toward lactogen receptors in Nb₂ cells and toward somatogen receptors in bovine liver decreased respectively to 30% and 20%. The present study indicates that out of the seven amino groups of human growth hormone, the ε-amino group of lysine-70 is excessively reactive toward FITC. Second, this particular amino group contributes to receptor binding and receptor activation. Lysine-70 is located in the loop between the first and second helix and close to the carboxy-terminal end of the first helix. This contribution is most likely the result of the formation of an electrostatic interaction between the hormone and the receptor. Fluorescein_Lys-70-hGH may therefore be utilized in further chemophysical studies, since the derivative largely preserves its binding and biological potency and the fluorescein moiety is located within the hormonal binding domain of GH to its biological receptor.