The reaction of tetranitromethane with pituitary, luteinizing and thyroid-stimulating hormones.

The reaction of tetranitromethane with the tyrosine residues of the pituitary glycoprotein hormones, luteinizing hormone (LH) and thyroid-stimulating hormone (TSH) has been compared. The total reaction products were inactive if sufficient reagent was used. Although polymer formation is a major reaction, nitrated LH monomer was obtained which was then separated into its a and /3 subunits and a fraction containing cross-linked monomer. The (Y subunit was fully nitrated at position 2 1; the two tyrosines at positions 92 and 93 were each partially nitrated while tyrosines 30 and 41 had not reacted. Nitrated LH-a recombined with either LH-0 or TSH-P as judged by gel electrophoresis, but only 10 to 25% of the hormonal activity expected after recombination of native subunits was restored. The immunological response of the nitrated products against antisera to the native hormone or to the subunits was unimpaired. The results show that nitration of tyrosine residues 21 and 92-93 of the a subunit does not interfere with its association to either LH-P or TSH-P, but it is probable that these residues are involved in the interaction of the two hormones with receptor structures at their respective target organs. The alternatives of dissociation in viva or of increased destruction in plasma as causes of loss of activity, however, are not excluded. The two tyrosines of LH-/3 isolated from the monomer were not nitrated although in much of the nitrated LH these residues participated in the formation of interchain or intramolecular cross-links. When isolated subunits are allowed to react with tetranitromethane under similar conditions, an additional tyrosine, residue 41, of the a chain is fully nitrated. The additional nitration of the o( chain partially or completely inhibits recombination with native LH-/?, nitrated LH-fit and native TSH-/3, thus indicating the participation of tyrosine 41 in the interaction between o( and p subunits. In LH-P nitration of tyrosine 59 and partial nitration of tyrosine 37 occurred, but the effects of nitration on the isolated /3 chain were less clear; some recombination and restoration of hormonal activity were found. Nitration of intact TSH, even in the presence of a limited amount of reagent, yielded largely polymeric material with a molecular weight range of 150,000 to 220,000, thus showing that some of the 16 tyrosines of its subunits are particu-