Magnetic Resonance Studies of the Interaction of Cupric Ion with Native and Modified Forms of Ribonuclease

摘要: Abstract The binding of copper to bovine pancreatic ribonuclease A, its carboxymethylated histidine-119 and histidine-12 derivatives, S S-protein, has been investigated at pH 5 6 by measuring the enhancement magnetic proton relaxation rate (PRR) water due bound copper. magnitude PRR factor is characteristic for site reflects protein environment. Titrations yield values constants number sites reveal interaction between sites. At 5, RNase A exhibit three independent sites, one strong with a dissociation constant, Kd, 7 x 10-4 m two weaker ones Kd approximately 8 10-3 m. 1-carboxymethylhistidine-119-RNase Sprotein have only weak, 3-carboxymethyl histidine-12-RNase site. site, ebb1, 6.1 almost same 1-carboxymethylhistidine119-RNase, 5.9, but significantly lower S, 5.1, suggesting more flexible structure first Cu(II)-binding Modification carboxymethylation results in drastic change environment this ebb1 = 30. To lesser extent, removal S-peptide also an increased value tight 9.0 S-protein. 6.0, patterns become much complex; all species except derivative cooperativity Cu(II) some association, rendering it impossible extract or eb individual from data. 3-Carboxylmethylhistidine-12-RNase exceptional that reveals equivalent noninteracting 6, equals 4 m, 24.3. Measurements as function temperature indicated chemical exchange ligands complexes so fast observed rates are determined dipolar correlation time hydration sphere Cu(II). energies activation 3 kcal per mole indicates rotational motion determines rate. inhibitor were respectively, cytidine2', 3'-cyclic phosphate substrate agreed within 2 less In electron paramagnetic resonance spectra solutions peaks corresponding free yielded those calculated data, indicating enhanced data accounted total bound. On basis other known properties tentatively assigned histidine-105 histidine-119.