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Methods of Biochemical Analysis: v. 30 (Methods of by David M. Glick

By David M. Glick

An encyclopaedia of tools in biochemical research, this contemporary sequence retains biochemists and analytical chemists abreast of experimental concepts and enhancements in biochemical strategies and instrumentation.

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In some cases, we might be able to sense the presence of the hydration layer not by its reluctance to hydrate a proton, but by its reflection of a proton coming from the bulk. That is what was measured in the inner space of a liposome. The proton dissociates at the normal rate, as in bulk water, but the space through which it can diffuse is smaller than the volume defined by the geometry of the liposome. We do observe a major discrepancy between the effective concentration of the proton and the predicted one.

The third rate constant that characterizes the dynamics of proton reaction with +O-* is the rate of proton escape out of the microcavity. This rate is most easily estimated from (-yl) the rate constant of the slow phase of (+OH*) fluorescent decay. During the second phase of decay, the reactants +OH*, +O-*, and H+ in the cavity are in apparent equilibrium (as averaged over the number of sites). Under such conditions, and the provisions that binding does not change the lifetime of the excited ligands, we can assume only two mechanisms that consume the N1population: (1) the irreversible decay of the excited state and (2) the irreversible loss of the proton to the buIk.

The dissociation can be prevented if the compound is dissolved in acid solution, pH < pK*, such as 2MHCl (line B). Under such conditions, we observe the emission of the neutral form with maximum at 445 nm. -. 450 QOH'HCI 500 nrn 550 Figure 12. Steady-state fluorescence emission of hydroxypyrene trisuIfonate. Fluorescence of 20pM hydroxypyrene trisulfonate. 0. Excitation at 400 nm. Fluorescence measured in arbitrary units at identical instrumental set up. 26 MENACHEM GUTMAN bands (line C) of equal intensity, typical for the anionic state (515 nm) and the neutral form, slightly shifted to shortened wavelength (435nm).

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