Forward quench experiment

Direct observation of the E p/t dNTP complex was obtained using pulse-chase experiments. In such experiments, incorporation of labeled nucleotide to an E p/t complex is either quenched by the addition of HC1 or allowed to proceed after the addition of a large excess of cold unlabeled dNTP (the chase step) followed by acid quench. In the HC1 quench experiments, the acid quenches all the enzyme-bound species. On the other hand, when the reaction is chased with cold dNTP, each of the enzyme-bound species is allowed to partition both in the forward and reverse directions. The amount of partitioning in the forward direction is observed as an excess of labeled product, compared with the acid quench experiment, while the dNTP that partitions in the reverse direction is diluted and remains unobservable. As an excess was observed and because the binding of dNTP to the E p/t complex is rapid, the observed flux or excess is mainly due to the E p/t dNTP complex. 

Pulse-chase/pulse-quench experiments with KF ° indicated accumulation of the nucleotide bound enzyme species, which would not be possible if the forward reaction was much faster than the rate of conformational closing. To explain this observation, the authors proposed the presence of a kinetic road block - a slow step after the phosphodiester bond formation. However, the results of the pulse-chase/ pulse-quench experiments can also be explained by designating chemistry as the slow step, meaning that the chemical step itself plays the role of the road block. The conclusion that chemistry is a fast step in the KF reaction pathway was made based on the observation of a small thio-efifect magnitude,which, as elaborated in the following section, should not be used as a solid evidence of the chemical step being nonrate limiting. 

Two mechanisms have been put forward to explain the photochemistry of chromium(in) complexes. Their photoreactivity has been ascribed to excitation either to the lowest spin-forbidden excited state, Eg, or to the lowest quartet excited states, and Mxg. Several years ago some quenching experiments on the photoaquation of the [Cr(NH3)2(NCS)4]- anion indicated that a quartet state was at least partially involved. Recently two papers concerning the photochemistry of the [Cr(CN)6] anion, chosen for the known large energy difference between the doublet and quartet states, have provided strong evidence for a quartet state as the photointermediale. Thus the observations that pyrazine and xanthone sensitize the photoaquation of [Cr(CN)e] , but that Michler s ketone and [Ru(bipy)3] + do not, rule out photoaquation via a doublet state. Likewise a comparison of phosphorescence and photolysis of solutions of [Cr(CN)6] in dimethylformamide showed that the photolysis, to [Cr(CN)5(DMF)] , could not proceed via the same excited state, Eg, as phosphorescence. Both of these investigations led to the implication of the... 

When the rate constants are such that the last term in Eq. (15) is not negligible, determination of the quantum yield of reaction as a function of sensitizer concentration will reveal back transfer this experiment also tests for sensitizer self-quenching (Section III.B). In the most frequently encountered case, forward transfer is much faster than back transfer (i.e., ke k.e) so that this term is small. The dependence of m on sensitizer concentration disappears and Eq. (15) reduces to the usual expression for quantum yield as a function of [A]. 

This chapter is intended to provide an overview of the results obtained from recent experimental studies of sensitized fluorescence and quenching of resonance radiation in alkali and mercury vapors, up to 1974. The many inconsistencies and discrepancies that still exist between experiment and theory as well as among the experimental results themselves, and that are a source of concern to those working in the field, will no doubt continue to be resolved as new ideas are put forward and as both theoretical and experimental techniques advance in precision and sophistication. 

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