Pro-Kineticist II - 2nd Order Global Analysis

Overview

Pro-K II builds substantially on the capabilities of our previous global analysis software, Pro-K, by enabling the 2^nd order global analysis of spectra-kinetic data gathered at multiple starting concentrations (previously only single spectra-kinetic data sets could be analysed). In addition Pro-KII can simultaneously analyse complimentary data sets gathered not only under different initial concentrations or pH's but can also combine different types of measurement, such as fluorescence and absorbtion spectra.

The key benefit of this is to significantly enhance the ability of the analysis to resolve reaction parameters. Rates and intermediate spectra, which were previously undefined, can now be determined.

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

Another 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 dependant data sets.

Another key addition to Pro-KII is support in the modelling of rapid equilibria a feature allows incorporation of rapid protonations and ligand binding steps defined by either fixed or variable equilibrium constants or, for protonations, group pK values. For example, this allows pH dependant data sets to be fully analysed by provision of the pK of both the protonation step as well as 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:

Buffer Equilibrium	B+H=BH	(pK fixed and B, H and BH all colourless)
Reaction	A+X<>C
	C+H=CH	(protonation equilibrium, pK known or fitted)
	CH>P

An important feature is that the buffering is fully modelled, i.e. it is not an approximation. The [H] can, and will, change during the reaction and this 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.