Experimental test of a method for determining causal connectivities of species in reactions

Abstract

Theoretical analysis has shown the possibility of determining causal connectivities of reacting species and the reaction mechanism in complex chemical and biochemical reaction systems by applying pulse changes of concentrations of one or more species, of arbitrary magnitude, and measuring the temporal response of as many species as possible. This method, limited to measured and pulsed species, is given here an experimental test on a part of glycolysis including the sequence of reactions from glucose to fructose 1,6-biphosphate, followed by the bifurcation of that sequence into two branches, one ending in glycerol 3-phosphate, the other in glyceraldehyde 3-phosphate. Pulses of concentrations of one species at a time are applied to the open system in a non-equilibrium stationary state, and the temporal responses in concentrations of six metabolites are measured by capillary zone electrophoresis. From the results of these measurements and the use of the theory for their interpretation, we establish the causal connectivities of the metabolites and thus the reaction mechanism, including the bifurcation of one chain of reactions into two. In this test case of the pulse method, no prior knowledge was assumed of the biochemistry of this system. We conclude that the pulse method is relatively simple and effective in determining reaction mechanisms in complex systems, including reactants, products, intermediates, and catalysts and their effectors. The method is likely to be useful for substantially more complex systems. For some time, we have studied and reported on the development of novel methods for the determination of reaction mechanisms in both theory and experiments. New methods are necessary to replace the guessing of reaction mechanisms, that is, the sequence of elementary reaction steps leading from reactants to products. Our approaches are designed not to dissect a system into its elementary steps to be studied one at a time, as has been practiced, but rather to keep the system, and hence all its interactions, intact. For a method for the determination of the mechanism of oscillatory reactions, chemical and biochemical, see ref. 1 (and references 9, 10, 14, 18, 38, and 41 therein); for the application of the techniques of genetic algorithms, see refs. 2 and 3; for general reactions, we have developed a method of correlation metric construction. Time-dependent correlations of the concentrations of two species at a time are transformed into correlation diagrams that show the reaction pathway and significant parts of the reaction mechanism (4, 5). This method was successfully tested with experiments on a part of glycolysis (6, 7). It can also yield predictions on the connectivities of reacting chemical species, but for that it requires more accurate measurements than necessary for the determination of reaction pathways.In yet another approach, we presented a theory of pulse perturbations for the determination of the connectivities of measured and pulsed chemical species in a reaction system (8) and, hence, the rea...

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