Time resolved photo-CIDNP

The role of Ca2+ in inducing refolding of a-lactalbumin is reflected in clean two-stage kinetics, with rate constants 6.0 and 1.3 s-1. The maximum concentration of the intermediate, monitored by stopped-flow fluorescence and time-resolved photo-CIDNP NMR, occurs at about 200 ms (327). 

Fig. 25. Time resolved CIDNP spectrum observed upon photo-induced electron transfer oxidation of hexamethyl-(Dewar)-benzene (0.02 M) by chloranil (0.02 M). The top spectrum was observed 5 ps after excitation with the frequency-tripled output (355 nm) of a Nd/YAG laser. The bottom spectrum was recorded in the dark...

The only pieces of hardware needed for photo-CIDNP are a light source and an unmodified NMR spectrometer. Pulsed lasers are most convenient for illumination, as they allow both time-resolved experiments (when the laser flash is followed by an acquisition pulse after a variable time delay) and steady-state ones (when the laser is triggered with a high repetition rate, thus providing quasi-continuous excitation). All the examples of this work draw on the second variant. Nevertheless, they yield kinetic information about much faster processes than would be observable by direct... 

Important mechanistic insights have been obtained in the last 5 years, particularly owing to the availability of such modern techniques as laser-flash time-resolved transient absorption spectroscopy [22,23], photo-CIDNP [21c, 24], and so on. It is thus the particular purpose of this review to analyze this recent work on mechanisms for photodecomposition of iodonium and sulfonium salts, and to evaluate its implications for practical applications of onium salt photochemistry. 

Figure 9 A time-resolved photo-CIDNP experiment. Further explanation, see text. Adapted from Ref. 75 with permission copyright (2006) Taylor 8t Francis Ltd, http / www.tandf.co.uk/journals.

Although the first case puts no demands on the energetics because escape is always a feasible process, no direct observation by a time-resolved CIDNP experiment seems to have been successful so far instead, it has repeatedly been reported that time-resolved photo-CIDNP experiments showed olefin radical cations or anions to be configurationally stable. The second and third cases are obviously only possible if the energy of the olefin triplet or the biradical lies below the energy of the radical ion pair. 

The determination of surface accessibilities in a protein is by now a well-established application of photo-ClDNP. Its operating principle is that a sensitizer in the bulk solution is photoexcited, forms a radical pair with an amino acid that is exposed to the solution, and so causes CIDNP to arise amino acids not accessible to the dye remain impolarized. To avoid disruptions of the structure the photore-action(s) must be cyclic. As this leads to exchange cancellation, one either observes the polarizations that remain because of relaxation in the free radicals or samples the geminate polarizations in a time-resolved CIDNP experiment. The latter appears preferable for quantitative conclusions as it also removes other artefacts, but cannot be applied to the pulse-labelling and related experiments described below. Commonly employed dyes are 2, 2 -dipyridyl 16 or flavins 17. As already mentioned in the preceding section, only tryptophan 10, tyrosine 11, and histidine 12 are polarizable. However, the reduction of the information content in a crowded protein spectrum by this selectivity is a much desired blessing rather than a drawback. 

Photochemically Induced Dynamic Nuclear Polarization (Photo-CIDNP) of Biological Molecules Using Continuous Wave and Time-Resolved Methods... 

Time-Resolved CIDNP. - Petrova et al , have used time-resolved photo-CIDNP in the dibenzyl-ketone-b-cyclodextrin inclusion complex. The photodecay of the inclusion complex generates a benzyl radical that is held in the cyclodextrin cavity for a time exceeding the nuclear spin relaxation time, from... 

Some features of the laser photo-CIDNP method will now be summarized. We have shown that hy employing a dye as sensitizer nuclear spin polarization can be generated in native proteins. When flavins are used as dyes nmr lines of Tyr, His, and Trp residues can be selectively enhanced when these residues are accessible to the photo-excited dye. Thus, the method constitutes a "surface probe" with the very high resolution of the nmr technique, which is capable of resolving lines due to individual residues. Large enhancements, often of the order of a magnitude or more, are translated in considerable time savings. 

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