Since these relaxation phenomena are time-dependent, kinetic information such as molecular motion is possible from the studies. More detailed treatments are available ( Abragam, 1973 and James, 1975). In the study of enzymes it is conceivable that a 1H spectrum of the enzyme can yield absorption peaks for each of the protons in the molecule. The two major click here problems with this NMR approach are the concentration of enzyme and resolution of the spectra. The signal-to-noise of the spectrum is directly proportional to

the concentration of the sample. Many enzymes may not be sufficiently soluble to yield a 1×10−3 M solution. Even if solubility is not a major problem, an increase in concentration increases the viscosity of the sample. In more viscous solutions rapid averaging of the sample no longer occurs and broad absorption lines are observed, which decreases resolution of the spectrum. In an enzyme of molecular Cetuximab weight approximately 70,000 (an average size protein) the rotational correlation time, τ, in aqueous solution may be estimated at 10−8 s using the Stokes–Einstein equation, assuming the protein is roughly globular. This enzyme is also expected to contain approximately 600 amino acids. The large number of residues results in a high number

of overlapping resonances because of the number of protons present. Although assignments of resonances of free amino acids ( Roberts and Jardetzky, 1970) and amino acids in small peptides have been made, the assignments of resonances which may be observed for an enzyme must be made for specific amino acid residues within the enzyme structure. This made a severe limitation in the past, and to solve this problem, an approach such as

specific amino acid derivatization prior to obtaining the spectrum often helped in making assignments. At present multi-pulse methods are used for structure determination. There is detailed information on peptides ( Wüthrich, 1986), and nuclear relaxation and Overhauser effects were successfully used in studies of enzyme substrate interactions ( Mildvan, 1989). The most useful approach to studying enzyme structure by protein NMR with a minimum almost of perturbation was the observation of the resonances from histidine. The C-2 and C-5 proton resonances are downfield from the aromatic protons (Markley, 1975). The classical use of these properties was with the small enzyme RNAase (Mr=23,500) Meadows and Jardetzky (1986) and the large enzyme (Mr=237,000) pyruvate kinase ( Meshitsuka et al., 1981). The C-2 proton resonance is especially sensitive to the ionization state of the imidazole nitrogens, thus the pKa for each individual histidine within the native enzyme can be obtained from titration studies.