On the use of tetranitromethane as a nitration reagent. The reaction of phenol side-chains in bovine and porcine trypsinogens and trypsins.

Abstract

Tetranitromethane, a reagent described by Riordan et al. in 1966 has been used for the nitration of tyrosines in bovine and porcine trypsinogen and trypsin. The reagent induces a great number of inter- and intra-molecular side-reactions. In particular, considerable polymerization due to crosslinking can be observed, especially with high concentrations of proteins. Monomers, dimers and polymers of higher degree can be easily separated by Sephadex G-100 chromatography. The existence of polymeric species has also been observed with ribonuclease, lysozyme and the Kunitz pancreatic inhibitor. 5 of the 10 tyrosines are sufficiently exposed at the surface of the trypsinogen molecule to be nitrated easily. The second-order kinetics of the chemical modification have been studied under different conditions and compared to the kinetics of nitration of N-acetyl-l-tyrosine ethyl ester. Nitration increases the bulk of the tyrosine side-chain and decreases the pK- of the phenol function from 10.2–10.3 to 6.9 in trypsinogen. This important modification of the properties of this residue has no effect on the catalytic activity of trypsin toward small synthetic ester or amide substrates but it decreases considerably the tryptic activity toward macromolecular substrates. For example, nitration of trypsin decreases by a factor of 15 the maximal rate of activation of chymotrypsinogen A; no change on Km has been observed.