Holoprotein formation of human chorionic gonadotropin: differential trace labeling with acetic anhydride

The effects of holoprotein formation in human CG (hCG) on the reactivities of several of the individual amino groups of each subunit were investigated by differential trace labeling with [3H]acetic anhydride. The alpha- and beta-subunits were labeled separately, as was hCG, under conditions chosen to ensure that an average of less than one amino group was modified per molecule. Although the beta-subunit contains fewer amino groups than the alpha-subunit, most of the 3H incorporation occurred in beta at the N-terminal region. Chemical and enzymatic cleavage of the subunits enabled us to identify several individual amino groups and, from measurements of the incorporated radioactivity of the free subunits and intact hormone, determine their protection factor, which is a measure of the reactivity and thus of the local environment and changes thereof upon holoprotein formation. Lys51 and Lys91 of alpha were approximately 2-fold more reactive and less reactive, respectively, in the alpha beta complex than in the free subunit. The alpha-amino group of alpha was characterized by comparable reactivities in the heterodimer and free subunit, as was Lys44/Lys45 when analyzed as a pair; the reactivity of alpha-Lys44 was slightly less in the holoprotein than in the free subunit. The alpha-amino group and Lys2 of beta could not be resolved by available cleavage procedures; consequently they were analyzed as a pair and found to be some 2-fold less reactive in the heterodimer than in the free subunit, as was Lys104 of beta. From these results, we can conclude that subunit assembly produces changes in the microenvironments of several amino groups, attributable to steric effects, specific intermolecular interactions, and localized conformational changes. Analysis of these data with reference to the recently determined crystal structure of hydrogen fluoride-treated hCG enabled a distinction to be made of these possibilities for several of the amino groups.