Studies with carbodiimide-cross-linked derivatives of bovine lutropin. I. The effects of specific group modifications on receptor site binding in testes.

The reaction of the water-soluble carbodiimide, lethyl-3(3-dimethylaminopropyl)carbodiimide, @DC) with bovine lutropin (bLH) has been studied. Increasing the concentration of EDC from 0.01 M to 0.1 M increased the extent of covalent cross-linking between the 1y and p subunits of bLH with a concomitant decrease in receptor-binding activity of the nondissociable product from 40% (0.01 M EDC) to 5% (0.1 M EDC) of control. The loss of biological activity of the crosslinked derivatives as measured by ability to stimulate testosterone production in dispersed interstitial cells paralleled the loss of receptor-binding activity, whereas immunological activity was less affected by the same modifications. Cross-linked bovine lutropin retains significant activity after exposure to conditions that rapidly dissociate native bLH into its constituent subunits. After 8 h incubation in 8 M urea at 37”C, cross-linked bLH retained about 50% of its initial receptor-binding activity, whereas native bLH retained only about 1% of its initial activity. Deamination of exposed lysine residues in native and cross-linked lutropin indicates that the latter contains 1 major intersubunit amide linkage. Amino acid analyses failed to detect any substantial tyrosine modification; further, treatment of cross-linked lutropin (LH) with hydroxylamine, which can reverse tyrosine modification, did not restore receptor-binding activity. Isoelectric focusing in polyacrylamide gels revealed that each cross-linked bLH preparation represents a population of derivatives with progressively more basic isoelectric points, suggesting substantial stable incorporation of carbodiimide into free acidic groups. In order to explore the possibility that the loss in activity of EDC-cross-linked derivatives prepared with greater than 0.01 M carbodiimide is due to carboxyl group modification, two related derivatives were prepared. Cross-linking was performed with 0.01 M EDC in the presence of 1 M methyl glycine ester and 1 M ethylene diamine with a resultant incorporation of an average of about six neutral and seven positively charged groups, respectively, into the free (negatively charged) acidic groups of the molecule. The purified derivative with neutral group substitution was only slightly less active than the corresponding derivative prepared in water (20 versus 40% of control activity), while partial replacement with a positively charged moiety drastically reduced receptor-binding activity