Simplification of the Proton Magnetic Resonance Spectrum of Ribonuclease by Difference Spectroscopy

Nuclear‐Magnetic‐Resonance Study of the Histidine Residues of S‐Peptide and S‐Protein and Kinetics of 1H‐2H Exchange of Ribonuclease A

“…It is noted that histidine-48 gives rise to a peak at pH 3.0 which, as previously observed [15,30,31], broadens and disappears above about pH 5.5. The amount of hydrogen-deuterium exchange increases with increasing times of incubation at 35 "C but this effect cannot be quantified because of the occurrence of a new peak X that also increases with time.…”

“…With RNase-A many workers [15,30,31,37] have observed the fact that in the absence of acetate ions the resonance from histidine-48 occurs at a constant chemical shift below about pH 5.4, but above this pH it rapidly broadens and disappears. Markley [37] has observed the reappearance of the resonance at higher field at pH > 6.1 and given an interpretation of the phenomenon.…”

Nuclear-Magnetic-Resonance-Spectroscopic Studies of the Amino Groups of Insulin

“…Spectral range was 4000 Hz. In a series of spectra collected at various pH values any particular spectrum was subtracted from the spectrum obtained at the next lowest pH and had substracted from is the spectrum obtained at the next highest pH value [4]. The spectra at 100 MHz were obtained on a continuous-wave Jeol model MH-100 'H NMR spectrometer at an ambient probe temperature of 26 "C using a capillary of tetramethylsilane in carbon tetrachloride as an external reference.…”

“…9) overlaps many other resonances in the insulin spectrum and it was not possible to make a specific assignment in the present spectra. The pH range over which the lysine-B29 8-methylene carbon resonance (2) lysine 6-C; (3) asparagine [K; (4) lysine & ; ( 5 ) leucine / K : (6) glycine a-C N-terminal protonated; (7) glycine cc-C non-terminal; (8) glycine cc-C N-terminal deprotonated; (9) proline 6-C; (10) alanine cc-C; (11) shifts to lower field is compatible with the pK values given in Table 1.…”

“…In particular, previously developed techniques for observation of methylated amino groups in proteins [2,3] have been applied to the study of the methylated amino groups of glycine-A 1, phenylalanine-B 1 and lysine-B29. In addition it is shown that application of 'H NMR difference spectroscopy [4] can be used to observe the methylene groups of lysine in unmodified proteins (see also [5]). The ability to observe the various insulin amino groups has led to a detailed analysis of the pH-dependent behaviour of the amino groups of glycine-Al, phenylalanine-B1 Abbreviation.…”