Complete tyrosine assignments in the high-field proton nuclear magnetic resonance spectrum of bovine pancreatic trypsin inhibitor selectively reduced and carboxamidomethylated at cystine 14-38

Abstract

The low-field portions of the 250-MHz 1H NMR spectra of native and chemically modified basic pancreatic trypsin inhibitor were studied as a function of pH over the range pH 5-13. In derivatives selectively reduced and carboxamidomethylated at cystine 14-38, resonances associated with 15 of the 16 protons of the aromatic rings of the 4 tyrosines of the inhibitor were located and assigned to specific tyrosyl residues. Titrations yielded pK''s for tyrosines 10, 21, 23 and 35 in the modified inhibitor of 9.9, 10.6, 11.6 and 11.0, respectively. Resonances associated with the 3 nitrotyrosine 10 protons of the mononitrated derivative and the 6 nitrotyrosine 10 and 21 protons of the dinitrated derivative were similarly located, assigned and titrated, yielding pK''s for nitrotyrosine 10 and 21 of 6.5 and 6.4, respectively. Previously reported results for derivatives with cystine 14-38 intact were revised on the basis of new data. Comparison of these revised results with the new data for derivatives with modified cystine 14-38 reveals no changes in pK''s for any tyrosine or nitrotyrosine ring and no changes in chemical shift for resonances of nitrotyrosine 21 or tyrosines 21 and 23. Modification of cystine 14-38 causes significant changes in chemical shifts of resonances of the nearby nitrotyrosine 10 and tyrosine and 10 and 35 rings. Tyrosine 35 remains relatively immobile, rotating less than 1600 times/s at 25.degree. C for pH''s in the range 5-13.