Direct observation of peptide exchange by stable isotope enrichment.
A method for the kinetic determination of peptide exchange using stable isotope enrichment is described. Synthetic 90% enriched (epsilon-13C)His 12 ribonuclease (RNase) (1-15) peptide was used as a probe to follow peptide exchange in the RNase S system by 13C nuclear magnetic resonance spectroscopy. The rate constant, k1, for dissociation of the RNase S complex containing the synthetic (1-15) peptide was found to be (4.1 +/- 0.3) x 10(-4) s-1 and its dissociation constant Kd, (0.2 +/- 0.8) x 10(-7) M, was greater than that of RNase S with natural S-peptide (residues 1 to 20) by a factor of five at 4 degrees C. This differences corresponds to the difference of the enthalpy of binding between the (1-15) and (1-20) peptides, which we determined to be 1.7 +/- 0.4 kcal/mol. This small enthalpy difference may originate from hydrogen bonding between Ser 16 and His 48 in the RNase S complex.
D002247:Carbon Isotopes; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D008433:Mathematics; D010455:Peptides; D011485:Protein Binding; D012260:Ribonucleases; D013816:Thermodynamics
D002247:Carbon Isotopes; D010455:Peptides; D012260:Ribonucleases