When two or more sites in an asymmetrical molecule may be involved in an equilibrium reaction there are more than one possible equilibrium constants. For example, the molecule -DOPA has two non-equivalent hydroxyl groups which may be deprotonated. Denoting -DOPA as LH, the following diagram shows all the species that may be formed (X = ).
The concentration of the species LH is equal to the sum of the concentrations of the two micro-species with the same chemical formula, labelled LH and LH. The constant K is for a reaction with these two micro-species as products, so that [LH] = [LH] + [LH] appears in the numerator, and it follows that this macro-constant is equal to the sum of the two micro-constants for the component reactions.
:K = k + k
However, the constant K is for a reaction with these two micro-species as reactants, and [LH] = [LH] + [LH] in the denominator, so that in this case
:1/K =1/ k + 1/k,
and therefore K =k k / (k + k).
Thus, in this example there are four micro-constants whose values are subject to two constraints; in consequence, only the two macro-constant values, for K and K can be derived from experimental data.
Micro-constant values can, in principle, be determined using a spectroscopic technique, such as infrared spectroscopy, where each micro-species gives a different signal. Methods which have been used to estimate micro-constant values include
* Chemical: blocking one of the sites, for example by methylation of a hydroxyl group, followed by determination of the equilibrium constant of the related molecule, from which the micro-constant value for the "parent" molecule may be estimated.
* Mathematical: applying numerical procedures to C NMR data.
Although the value of a micro-constant cannot be determined from experimental data, site occupancy, which is proportional to the micro-constant value, can be very important for biological activity. Therefore, various methods have been developed for estimating micro-constant values. For example, the isomerization constant for -DOPA has been estimated to have a value of 0.9, so the micro-species LH and LH have almost equal concentrations at all pH values.