Pro-Kineticist II

2nd Order Global Analysis

Overview

Pro-KII builds substantially on the capabilities of our previous global analysis software, Pro-K, by enabling the 2nd-order global analysis of spectra-kinetic data gathered at different starting concentrations. In addition, Pro-KII can simultaneously analyse complementary data sets from different types of measurement, such as fluorescence and absorption spectra. The key benefit of Pro-KII is to better resolve reaction parameters: rates and intermediate spectra, which were previously undefined, can now be determined.

The simplest example illustrating the 2nd-order advantage is in solving a second order binding reaction: A+B>C. With 1st-order global analysis, the spectra of A and B cannot be resolved unless the spectrum of one of the reactants is predetermined. However, by performing the reaction at two (or more) starting concentrations of A and B and submitting all the data to a 2nd-order analysis in Pro-KII, the individual spectra can be resolved because the extra information is sufficient to define a unique solution.

A more complex system which can now be successfully determined is the coupled equilibrium A+B<>C<>D. All spectra and forward and reverse rates can be calculated by provision of a suitable combination of concentration dependent data sets.

Pro-KII supports the modelling of rapid equilibria, a feature that allows incorporation of rapid protonation and ligand binding steps defined by fixed or variable equilibrium constants or, for protonation, group pK values. For example, it allows pH-dependent data sets to be fully analysed by provision of the pK of both the protonation step and any buffers present in the system. In these systems, a fixed pK for the buffer is usually provided (with colourless reactants and products). The protonation step in the chemistry may also represented by group pK that can then become an optionally fitted parameter.

The model would be represented as:

An important feature is that the buffering is fully modelled, i.e. it is not an approximation. The proton concentration can, and will, change during the reaction and is fully modelled by the algorithm. This is significant in that it is no longer necessary to experiment in strongly buffered environments, as pH changes can now be allowed during the reaction as all changes are fully modelled through inclusion of all relevant equilibria.