Preparation, specific binding, and biological activity of a monosubstituted fluorescein derivative of prolactin

A monosubstituted fluorescein derivative of ovine prolactin has been prepared by the reaction of fluorescein isothiocyanate with the hormone at pH 8.0. Reaction under the specified conditions results in a derivative incorporating one fluorescein moiety per molecule of prolactin as judged by coordinated UV spectroscopy and amino acid analysis. The absence of NH2-terminal threonine in the derivative indicates that the point of fluorescein attachment is the NH2-terminal amino group, whose substitution is expected to be favored at pH 8.0. The derivative was found to retain significant specific binding on both rabbit mammary and rat ventral prostate membrane preparations in competition with 125I-labeled prolactin, and to show saturable stimulation of progesterone production in primary cultures of rat granulosa cells. It is anticipated that this derivative will be useful in studies of prolactin-target cell interactions. This study shows that NH2-terminal monosubstituted derivatives of ovine prolactin are readily formed under specified conditions at pH 8.0; these conditions may be useful in the preparation of other derivatives of this type.