Isotopic labeling, slow exchange

The first experiment to be recorded on isotope-labeled proteins is the [ N/HJ-HSQC experiment. Inspection of the [ N. HJ-correlalion map and simple counting of cross peaks reveals whether multiple conformers exist, whether some parts of the backbone signals are broadened, possibly because of slow conformational exchange, or whether parts of the sequence are not visible at all. As mentioned above, the spectrum will also show whether the protein is well folded or not. 

B. Slow exchange studied by isotopic labeling technique... 

B. Slow Exchange Studied by Isotopic Labeling Technique... 

To study water exchange on aqua metal ions with very slow exchange of water molecules, an isotopic labeling technique using oxygen-17 can be used. A necessary condition for the applicability of this technique is that the life time, tm, of a water molecule in the first coordination shell of the ion is much longer that the time needed to acquire the 0-NMR spectrum. With modern NMR spectrometers and using enrichments up to 40% in the acquisition time can be as short as 1 s. 

By stirring the normal l6C) compound with a large excess of isotopically labelled water, H2l80, for a few hours in the presence of a drop of acid they were able to make the required labelled compound. Without the acid catalyst, the exchange is very slow. Acid catalysis speeds the reaction up by making the carbonyl group more electrophilic so that equilibrium is reached more quickly. The equilibrium is controlled by mass action—180 is in large excess. 

There is no scrambling of the isotopically-labelled water between metal ions all water molecules remains completely with the one metal centre throughout the reaction, because the rate at which water is exchanged with bulk water is very slow compared with the rate at which the electron transfer reaction occurs. The reaction is observed to be first order with respect to both reactants, consistent with a bimolecular encounter process. 

A number of hydrated cations in aqueous solution undergo exchange with the solvent at rates slow enough to be observed on the NMR spectroscopic timescale by using isotopic labelling has / = f, while both and are NMR inactive. Different chemical shifts are observed for the nuclei in bulk and coordinated water, and from the signal intensity ratios, hydration numbers can be obtained. For example, Al + has been shown to be present as [A1(H20)6] +. 

Johnston (1956) and Johnston and Manno (1957) pointed out that the detection ability of modern low-level counting extends the range of measurable kinetics to chemical half-times of millions of years or to the limit of radiation-induced reaction. Johnston and Manno (1957) used the isotopic technique to study the slow exchange between iodobenzene and potassium iodide-131 over the range 16-46°C. The reaction was shown to have a half-life up to 400 years. Conway and Libby (1958) used carboxyPKl-labeled alanine to study its rate of decarboxylation at temperatures ranging from 373-1 to 426-6°K and determined half lives for the reaction under various conditions as high as ten billion years. 

Although HSOs" is slow to exchange O atoms with water (ti/2 1 h) in solution, an isotope exchange is missed for HSOs" coordinated on a hypothesized intermediate of di-/x-oxo Mn2 dimer. Their isotope-labeling experiment is consequently incomplete, so that it could not be excluded that the O atom comes from the oxidant (ClO or HSOs"). They proposed the catalytic cycle for O2 evolution in Fig. 10, which involves a hypothesized intermediate of di-... 

---