# Predicting color of a TMB derivative affected by peroxidase

The ELISA test on HIV works by verifying the presence of peroxidase within samples. The verification process is done through the enzymatic reaction that said peroxidase has on TMB (3,3',5,5'-tetramethylbenzidine) in an environment that contains $$\ce{H2O2}$$. This reaction leaves the enzyme untouched and transforms the $$\ce{H2O2}$$ into $$\ce{2H2O}$$ by adding two hydrogen atoms taken from the TMB. This also transforms the TMB into another compound, 4-(4-imino-3,5-dimethylcyclohexa-2,5-dien-1-yl)-2,6-dimethylcyclohexa-2,5-dien-1-imine, which has a blue color in that specific case.

Therefore my question is, how could one predict from the structure of the one product that it is going to have a blue color in that situation? I know that it probably comes from the conjugated double bonds but how do their count relate to the absorbed wavelength(s)?

$$\begin{array}{l|cr} \text{Substance} & \lambda_\mathrm{max}, \ \pu{nm} & \epsilon, \ \pu{L mol-1 cm-1} \\\hline \ce{CH2=CH2} & \pu{180nm} & 10000 \\ \ce{CH2=CH-CH=CH2} & \pu{217nm} & 21000 \\ \text{Vitamin A (5 conjugate double bonds)} & \pu{328nm} & 51000 \\ \text{Benzene} & \pu{255nm} & 180 \\ \text{Naphthalene} & \pu{286nm} & 360 \\ \text{Anthracene} & \pu{325nm} & 7100 \\ \hline \end{array}\\ \epsilon = \text{Molar Absorption Coefficient}; \ \lambda_\mathrm{max} = \text{Wavelength of maximum absorption peak}$$
Thus, one can suggest diimine, 4-(4-imino-3,5-dimethylcyclohexa-2,5-dien-1-yl)-2,6-dimethylcyclohexa-2,5-dien-1-imine, which contains 5 directly conjugated double bonds (two of them are imines), may absorb wavelengths of lights way larger than $$\pu{328 nm}$$ given for Vitamin A. That's because it involves transition of $$n \rightarrow \pi^*$$ in addition to $$\pi \rightarrow \pi^*$$ in Vitamin A (e.g., $$\pi \rightarrow \pi^*$$ in acetone is $$\pu{195 nm}$$ in addition to $$n \rightarrow \pi^*$$ transition at $$\pu{274 nm}$$, when compared to $$\pi \rightarrow \pi^*$$ transition of ethylene at $$\pu{180 nm}$$). Experimental evidence has shown that 4-(4-imino-3,5-dimethylcyclohexa-2,5-dien-1-yl)-2,6-dimethylcyclohexa-2,5-dien-1-imine absorbs light at $$\pu{450 nm}$$, giving its characteristic complimentary color $$\color{blue}{\text{Blue}}$$ (Ref.1).