HUMAN PITUITARY GROWTH HORMONE . STUDIES OF TRYPTOPHAN RESIDUE

The single tryptophan residue in the human growth hormone molecule has been quantitatively and specifically reacted with 2-nitrophenylsulfenyl chloride and 2-hydroxy-5-nitrobenzyl bromide using 50% acetic acid as reaction media. The derivatives have been characterized by biological, chemical, and biophysical techniques. The nitrophenylsulfenyl derivative (I) retains full growth-promoting activity and its physicochemical properties are similar to those of the native molecule. The monolabeled 2-hydroxy-5-nitrobenzyl derivative (II) possesses much less growth-promoting potency, while the doubly labeled derivative (III) is almost completely devoid of this biological activity. The relative looseness of the molecular structure and the lowered stability of these latter two derivatives (II and III) is shown by the higher rate of tryptic digestion and the irreversibility of their spectrophotometric titration in aqueous media as compared with the native hormone. When tested for lactogenic activity, all these derivatives were inactive. It may therefore be concluded that the tryptophan residue in the human growth hormone molecule is not essential for growth-promoting activity, but, in contrast, may play an important role in the lactogenic activity of the hormone, suggesting that there are two different “active sites” for the two biological activities. The fact that the phenolic groups of the 2-hydroxy-5-nitrobenzyl moieties in derivatives II and III have an abnormal ionization behavior, and that total alkylation of the tryptophan residue occurs in 50% acetic acid, but not in 0.2 m acetic acid solution suggested that the tryptophan residue is “buried” in the interior of the hormone molecule, and is exposed in 50% acetic acid. © 1969, American Chemical Society. All rights reserved.