CHARACTERIZATION OF THE INTERACTION BETWEEN BOVINE PANCREATIC TRYPSIN-INHIBITOR AND THIOCYANATE BY NMR

The interaction between Bovine Pancreatic Trypsin Inhibitor and thiocy anate was studied using NMR spectroscopy following several experimenta l approaches. The chemical shift variations of the BPTI protons in the absence and in the presence of increasing thiocyanate concentrations (up to 0.2 M) were significant (> 0.05 ppm) for 30 protein protons bel onging to 20 residues. The largest deviation, 0.2 ppm, was observed fo r the amide backbone proton of Arg42 in the absence of thiocyanate and in the presence of 40 molar equivalents of thiocyanate. The influence of the presence of thiocyanate on the electrostatic potential surroun ding the protein was demonstrated by NOESY spectra selective at the wa ter frequency: the presence of SCN- favours acid catalysed exchange an d disfavours base catalysis. However, a specific effect of thiocyanate was pointed out since the comparison of the chemical shifts in the pr esence of 40 molar equivalents of KSCN and KCl, respectively, showed m uch more as well as larger deviations compared to measurements in the absence of salt. A dissociation constant, K-D, for a 1/1 complex betwe en BPTI and thiocyanate was calculated from chemical shifts measuremen ts: K-D = 89 +/- 8 mM. A second value, K-D = 99 +/- 10 mM, was extract ed from (SCN)-N-15 relaxation time measurements. (C) 1998 Elsevier Sci ence B.V. All rights reserved.